27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552, and 65577 molecules and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules, designated 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 and 65577 nucleic acid molecules. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 and 65577 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene has been introduced or disrupted. The invention still further provides isolated 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteins, fusion proteins, antigenic peptides and anti-27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 antibodies. Diagnostic and therapeutic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 10/391,364, filed Mar. 18, 2003 now U.S. Pat. No. 7,094,587, whichis a continuation-in-part of U.S. patent application Ser. No.09/950,370, filed Sep. 10, 2001 (abandoned), which claims the benefit ofU.S. Provisional Application Ser. No. 60/231,084, filed Sep. 8, 2000.U.S. patent application Ser. No. 10/391,364 is also acontinuation-in-part of U.S. patent application Ser. No. 10/294,039,filed Nov. 13, 2002 (abandoned), which claims the benefit of U.S.Provisional Application Ser. No. 60/338,587, filed Nov. 13, 2001. U.S.patent application Ser. No. 10/391,364 is also a continuation-in-part ofU.S. patent application Ser. No. 10/266,035, filed Oct. 7, 2002(abandoned), which claims the benefit of U.S. Provisional ApplicationSer. No. 60/328,198, filed Oct. 9, 2001. U.S. patent application Ser.No. 10/391,364 is also a continuation-in-part of U.S. patent applicationSer. No. 09/717,926, filed Nov. 21, 2000 now U.S. Pat. No. 6,569,657,which claims the benefit of U.S. Provisional Application Ser. No.60/214,707, filed Jun. 27, 2000. U.S. patent application Ser. No.10/391,364 is also a continuation-in-part of U.S. patent applicationSer. No. 10/268,036, filed Oct. 9, 2002 (abandoned), which claims thebenefit of U.S. Provisional Application Ser. No. 60/327,820, filed Oct.9, 2001. U.S. patent application Ser. No. 10/391,364 is also acontinuation-in-part of U.S. patent application Ser. No. 09/922,138,filed Aug. 3, 2001 (abandoned), which claims the benefit of U.S.Provisional Application Ser. No. 60/229,299, filed Sep. 1, 2000. U.S.patent application Ser. No. 10/391,364 is also a continuation-in-part ofU.S. patent application Ser. No. 09/945,327, filed Aug. 31, 2001(abandoned), which claims the benefit of U.S. Provisional ApplicationSer. No. 60/229,425, filed Aug. 31, 2000. U.S. patent application Ser.No. 10/391,364 is also a continuation-in-part of U.S. patent applicationSer. No. 10/163,316, filed Jun. 5, 2002 (abandoned), which claims thebenefit of U.S. Provisional Application Ser. No. 60/297,863, filed Jun.13, 2001. U.S. patent application Ser. No. 10/391,364 is also acontinuation-in-part of U.S. patent application Ser. No. 10/103,377,filed Mar. 21, 2002 (abandoned), which claims the benefit of U.S.Provisional Application Ser. No. 60/278,347, filed Mar. 23, 2001. Theentire contents of each of the above-referenced patent applications areincorporated herein by this reference.

BACKGROUND OF THE INVENTION

The enormous variety of biochemical reactions that comprise life arenearly all mediated by a series of biological catalysts known asenzymes. Enzymes are proteins which possess specific catalyticactivities that enable them to catalyze a series of reactions, henceenabling metabolic pathways to degrade and to reconstruct productsneeded to maintain organisms. By the binding of substrates throughgeometrically and physically complementary reactions, enzymes arestereospecific in binding substrates as well as in catalyzing reactions.The stringency for this stereospecificity varies as some enzymes aremore specific to the identity of their substrates, while others arecapable of binding multiple substrates and can catalyze numerous typesof reactions.

Examples of enzymes include, for example, phospholipases, serinecarboxypeptidases, trypsin-like serine proteases, aldehydedehydrogenases, ubiquitin-protein ligases, protein kinases, hydrolasesand matrix metalloproteinases. Such enzymes have the ability to, forexample: to reversibly phosphorylate proteins in order to regulateprotein activity in eukaryotic cells; to catalyze the hydrolysis of anacyl or phosphoacyl bond of a phospholipids; to modulate removal ofCOOH-terminal residues, i.e., having carboxypeptidase activity; tomodulate the transfer of an acyl group from a donor to an acceptormolecule, i.e., having acyltransferase activity; to degrade proteins; tophosphorylate carbohydrates; to oxidate an aldehyde; to modulateubiquitination of a substrate, e.g., a protein targeted for degradation;to modulate substrate specificity for ubiquitination; to reversiblyphosphorylate proteins in order to regulate protein activity ineukaryotic cells; to interact with cytotoxins and metabolites (e.g.,lactoylglutathione, a glutathione-conjugated metabolite, ahydroxycarboxylic acid, and the like); to catalyze the metobolism of acytotoxin or metabolite; to hydrolyze a thioester containing compound(e.g., lactoylglutathione, and the like); to catalyze the formation of athioester conjugation on a substrate (e.g., lactate or ahydroxycarboxylic acid); to cleave or modulate the degredation ofproteins or peptides of the extracellular matrix; to catalyze ormodulate catalysis of cleavage of covalent bonds within or between aminoacid residues (e.g., in extracellular matrix, cell-surface, andextracellular proteins); as well as many others. Accordingly, thereexists a need to identify additional human enzymes, for example, for useas disease markers and as targets for identifying various therapeuticmodulators.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel nucleic acid molecules and proteins encoded by such nucleic acidmolecules, referred to herein as “27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577”. The 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 nucleic acidand protein molecules of the present invention are useful as modulatingagents in regulating a variety of cellular processes, e.g., including,but not limited to cell proliferation, differentiation, growth anddivision. In particular, these nucleic acid molecules will beadvantageous in the regulation of any cellular function, uncontrolledproliferation and differentiation, such as in cases of cancer.Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 proteins or biologically active portionsthereof, as well as nucleic acid fragments suitable as primers orhybridization probes for the detection of 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577-encoding nucleicacids.

The nucleotide sequence of the cDNA encoding 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577, and the amino acidsequence of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 polypeptides are depicted in Table 1.

TABLE 1 Sequences of the invention ATCC accession Gene Name cDNA ProteinCoding Region number 27877 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 and 6PTA-3217 and 4 and 5 18080 SEQ ID NO: 11 SEQ ID NO: 12 SEQ ID NO: 13 N/A14081 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 N/A 32140 SEQ ID NO: 25SEQ ID NO: 26 SEQ ID NO: 27 PTA-3424 50352 SEQ ID NO: 29 SEQ ID NO: 30SEQ ID NO: 31 N/A 16658 SEQ ID NO: 35 SEQ ID NO: 36 SEQ ID NO: 37 N/A14223 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 40 N/A 16002 SEQ ID NO: 41SEQ ID NO: 42 SEQ ID NO: 43 N/A 50566 SEQ ID NO: 73 SEQ ID NO: 74 SEQ IDNO: 75 N/A 65552 SEQ ID NO: 76 SEQ ID NO: 77 SEQ ID NO: 78 N/A 65577 SEQID NO: 86 SEQ ID NO: 87 SEQ ID NO: 88 N/A

Accordingly, in one aspect, the invention features a nucleic acidmolecule which encodes a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein or polypeptide, e.g., abiologically active portion of the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein. In a preferredembodiment, the isolated nucleic acid molecule encodes a polypeptidehaving the amino acid sequence of SEQ ID NO:2, 5, 12, 21, 26, 30, 36,39, 42, 74, 77 or 87. In other embodiments, the invention providesisolated 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 nucleic acid molecules having the nucleotide sequenceshown in SEQ ID NO:1, 3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37,38, 40, 41, 43, 73, 75, 76, 78, 86 or 88 or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC Accession NumberPTA-3217 or PTA-3424. In still other embodiments, the invention providesnucleic acid molecules that are substantially identical (e.g., naturallyoccurring allelic variants) to the nucleotide sequence shown in SEQ IDNO:1, 3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43,73, 75, 76, 78, 86 or 88 or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC Accession Number PTA-3217 or PTA-3424.In other embodiments, the invention provides a nucleic acid moleculewhich hybridizes under a stringent hybridization condition as describedherein to a nucleic acid molecule comprising the nucleotide sequence ofSEQ ID NO:1, 3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37, 38, 40,41, 43, 73, 75, 76, 78, 86 or 88 or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC Accession Number PTA-3217 orPTA-3424, wherein the nucleic acid encodes a full length 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteinor an active fragment thereof.

In a related aspect, the invention further provides nucleic acidconstructs which include a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 nucleic acid molecule describedherein. In certain embodiments, the nucleic acid molecules of theinvention are operatively linked to native or heterologous regulatorysequences. Also included are vectors and host cells containing the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 nucleic acid molecules of the invention e.g., vectors and hostcells suitable for producing polypeptides.

In another related aspect, the invention provides nucleic acid fragmentssuitable as primers or hybridization probes for the detection of 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577-encoding nucleic acids.

In still another related aspect, isolated nucleic acid molecules thatare antisense to a 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 encoding nucleic acid molecule areprovided.

In another aspect, the invention features 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptides, andbiologically active or antigenic fragments thereof that are useful,e.g., as reagents or targets in assays applicable to treatment anddiagnosis of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577-associated disorders. In another embodiment, theinvention provides 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 polypeptides having a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 activity.

In other embodiments, the invention provides 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptides, e.g., a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 polypeptide having the amino acid sequence shown in SEQ ID NO:2,5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87 or the amino acid sequenceencoded by the cDNA insert of the plasmid deposited with ATCC AccessionNumber PTA-3217 or PTA-3424; an amino acid sequence that issubstantially identical to the amino acid sequence shown in SEQ ID NO:2,5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87 or the amino acid sequenceencoded by the cDNA insert of the plasmid deposited with ATCC AccessionNumber PTA-3217 or PTA-3424; or an amino acid sequence encoded by anucleic acid molecule having a nucleotide sequence which hybridizesunder a stringent hybridization condition as described herein to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75,76, 78, 86 or 88 or the nucleotide sequence of the insert of the plasmiddeposited with ATCC Accession Number PTA-3217 or PTA-3424, wherein thenucleic acid encodes a full length 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein or an active fragmentthereof.

In a related aspect, the invention further provides nucleic acidconstructs which include a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 nucleic acid molecule describedherein.

In a related aspect, the invention provides 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptides orfragments operatively linked to non-27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 polypeptides to form fusionproteins.

In another aspect, the invention features antibodies and antigen-bindingfragments thereof, that react with, or more preferably specifically orselectively bind 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 polypeptides.

In another aspect, the invention provides methods of screening forcompounds that modulate the expression or activity of the 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577polypeptides or nucleic acids.

In still another aspect, the invention provides a process for modulating27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 polypeptide or nucleic acid expression or activity, e.g., usingthe compounds identified in the screens described herein. In certainembodiments, the methods involve treatment of conditions related toaberrant activity or expression of the 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptides ornucleic acids, such as conditions or disorders involving aberrant ordeficient 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 expression. Examples of such disorders include, but arenot limited to cellular proliferative and/or differentiative disorders,brain disorders, blood vessel disorders, platelet disorders, breastdisorders, colon disorders, kidney disorders, lung disorders, ovariandisorders, prostate disorders, hematopoeitic disorders, pancreaticdisorders, skeletal muscle disorders, testicular disorders, skindisorders, hormonal disorders, disorders associated with bonemetabolism, immune e.g., inflammatory, disorders, cardiovasculardisorders, endothelial cell disorders, liver disorders, viral diseases,pain, metabolic disorders, anemias, angiogenesis, neoplastic disorders,endocrine disorders, neurological disorders and heart disorders.

The invention also provides assays for determining the activity of orthe presence or absence of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 polypeptides or nucleic acidmolecules in a biological sample, including for disease diagnosis.

In a further aspect, the invention provides assays for determining thepresence or absence of a genetic alteration in a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptide ornucleic acid molecule, including for disease diagnosis.

In another aspect, the invention features a two dimensional array havinga plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence. At least one address of the pluralityhas a capture probe that recognizes a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 molecule. In one embodiment,the capture probe is a nucleic acid, e.g., a probe complementary to a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 nucleic acid sequence. In another embodiment, the capture probe isa polypeptide, e.g., an antibody specific for 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptides.Also featured is a method of analyzing a sample by contacting the sampleto the aforementioned array and detecting binding of the sample to thearray.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

Human 27877

The invention relates to a novel phospholipase protein that can exist inat least two forms, herein designated the ‘short’ and ‘long’ forms. Thephospholipase (i.e., in either form) is referred to herein as “27877,”and can exhibit phospholipase A1 activity. Phosphatidic acids arepreferred substrates for the catalytic activity of 27877, althoughnon-phosphatidic acid phospholipids can also act as substrates for theenzyme.

The human short form of 27877 sequence (SEQ ID NO:1), which isapproximately 2981 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 2622nucleotides (nucleotides 202-2823 of SEQ ID NO:1; nucleotides 1-2622 ofSEQ ID NO:3), not including the terminal codon. The coding sequenceencodes a 874 amino acid protein (SEQ ID NO:2).

The long form human 27877 cDNA sequence (SEQ ID NO:4), which isapproximately 3065 nucleotide residues long including non-translatedregions, contains a predicted methionine-initiated coding sequence ofabout 2706 nucleotide residues, excluding termination codon (i.e.,nucleotide residues 202-2907 of SEQ ID NO:4; nucleotides 1-2706 SEQ IDNO:6). The coding sequence encodes a 902 amino acid protein (SEQ IDNO:5).

Human 27877 proteins contain a predicted transmembrane domain at aboutamino acid residues 620-636 of SEQ ID NOs:2 and 5. 27877 proteins canthus exist in a membrane bound form comprising two extra-membranedomains (the first corresponding to about amino acid residues 1-619 ofeach of SEQ ID NOs:2 and 5, and the second corresponding to about aminoacid residues 637 through the carboxyl terminus of each of thesesequences) separated by a transmembrane domain. In one embodiment, a27877 protein can exist in a form in which the region corresponding toabout amino acid residues 1-619 of each of SEQ ID NOs:2 and 5 isoriented on the non-lumenal side of a membrane (e.g., a cell membrane ora nuclear or other organellar membrane) and the region corresponding toabout amino acid residues 637 through the carboxyl terminus of each ofSEQ ID NOs:2 and 5 is oriented on the lumenal side of the membrane. Inanother embodiment, the regions have the opposite orientations. In stillanother embodiment, the 27877 protein exists in a soluble form (i.e.,not inserted in a membrane).

The human 27877 protein has predicted N-glycosylation sites (Pfamaccession number PS00001) at about amino acid residues 486-489 and690-693 of each of SEQ ID NOs:2 and 5; a predicted cAMP-/cGMP-dependentprotein kinase phosphorylation site (Pfam accession number PS00004) atabout amino acid residues 145-148 of each of SEQ ID NOs:2 and 5;predicted protein kinase C phosphorylation sites (Pfam accession numberPS00005) at about amino acid residues 8-10, 99-101, 180-182, 189-191,343-345, 367-369, 375-377, 588-590, 733-735, and 778-780 of each of SEQID NOs:2 and 5; predicted casein kinase II phosphorylation sites (Pfamaccession number PS00006) located at about amino acid residues 92-95,104-107, 141-144, 226-229, 244-247, 271-274, 271-274, 355-358, 399-402,450-453, 488-491, 575-578, 713-716, 778-781, 782-785, 813-816, and846-849 of SEQ ID NO:2 (about amino acid residues 92-95, 104-107,141-144, 226-229, 244-247, 271-274, 271-274, 355-358, 399-402, 450-453,488-491, 575-578, 713-716, 778-781, 782-785, and 874-877 of SEQ IDNO:5); a predicted tyrosine kinase phosphorylation site (Pfam accessionnumber PS00007) at about amino acid residues 579-586 of each of SEQ IDNOs:2 and 5; and predicted N-myristoylation sites (Pfam accession numberPS00008) at about amino acid residues 18-23, 35-40, 109-114, 133-138,139-144, 151-156, 530-535, 541-546, 601-606, 624-629, 635-640, 761-766,and 828-833 of SEQ ID NO:2 (about amino acid residues 18-23, 35-40,109-114, 133-138, 139-144, 151-156,530-535, 541-546, 601-606, 624-629,635-640, 761-766, and 856-861 of SEQ ID NO:5).

For general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997,Protein 28:405-420).

A plasmid containing the nucleotide sequence encoding the short form ofhuman 27877 was deposited with American Type Culture Collection (ATCC®),10801 University Boulevard, Manassas, Va. 20110-2209, on Mar. 23, 2001and assigned accession number PTA-3217. A plasmid containing thenucleotide sequence encoding the short form of human 27877 was depositedwith ATCC® on Mar. 23, 2001 and assigned accession number PTA-3217.These deposits will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. These deposits were made merely as aconvenience for those of skill in the art and are not an admission thata deposit is required pursuant to 35 U.S.C. § 112.

Phospholipids (sometimes designated ‘phosphatides’) are mixed esters offatty acids and phosphoric acid with an alcohol such as glycerol orsphingosine. Glycerol-based phospholipids have a phosphoryl moietyesterified with a hydroxyl moiety at one end of the glycerol moiety andat least one fatty acyl moiety esterified with the remaining glyceroylhydroxyl moieties. The phosphoryl moiety can be esterified with anadditional moiety (e.g., choline, ethanolamine, serine, inositol or aninositol phosphate), some of which (e.g., choline orinositol-4-phosphate) can have a charged moiety. Phospholipids are theprimary lipid component of most animal cellular membranes. Most membranephospholipids comprise two fatty acyl moieties, often including both asaturated fatty acyl moiety and an unsaturated fatty acyl moiety.

Phospholipases are a ubiquitous class of enzymes that catalyzehydrolysis of phospholipids. Multiple classes of phospholipases areknown, and phospholipases can be classified based on the phospholipidbond of which the enzyme catalyzes hydrolysis. For example,phospholipase A1 enzymes cleave the acyl moiety esterified at theglyceroyl hydroxyl moiety most distal from the phosphoryl moiety in aphospholipid. One known phospholipase A1 enzyme is designatedphosphatidic acid-preferring phospholipase A1 and was isolated frombovine testis (Higgs et al., 1998, J. Biol. Chem. 273:5468-5477).Phospholipase A2 enzymes more specifically cleave the acyl moiety at theglyceroyl hydroxyl moiety adjacent the phosphoryl moiety of aphospholipid. Phospholipase B enzymes can cleave acyl moieties fromeither of these positions. Phospholipase C enzymes cleave the phosphorylmoiety from the glycerol backbone of a glycerophosphatide, andphospholipase D enzymes can cleave phosphatidic acid from a moiety boundwith the phosphoryl moiety.

Because hydrolyzed phospholipids and products generated therefrom (e.g.,arachidonic acid and lipoxygenase- and cyclooxygenase-catalyzed reactionproducts such as prostaglandins) can act as second messengers incellular signaling systems, expression of many phospholipases is highlyregulated in cells. Many and complex array of regulatory mechanisms havebeen described for regulating phospholipase expression, some involvingcytoplasmic proteins, notably G-proteins, as well as different effectorlipids (e.g., phosphatidylinositol-4,5-biphosphate) or Ca²⁺.Phospholipase expression can be modulated by numerous signaltransduction pathways, and phospholipases can also participate innumerous signal transduction pathways.

Numerous phospholipases have been described. However, in view of thewidespread and critical nature of phospholipase activities in normal andpathological physiological processes, a need exists for identificationof further members of this protein family. The present inventionsatisfies this need by providing a novel human phospholipase.

The 27877 protein contains a significant number of structuralcharacteristics in common with members of the phospholipase family. Onecharacteristic of the lipase family of enzymes is the presence of fiveamino acids that have the consensus sequence Xaa₁-Xaa₂-Ser-Xaa₃-Gly (SEQID NO:10), wherein Xaa₁ is Gly or Ser and each of Xaa₂ and Xaa₃ can beany amino acid residue. This sequence is highly conserved among lipasesand contains the active serine nucleophile present in most lipases. Thissequence is present in the 27877 amino acid sequence asSer-His-Ser-Leu-Gly (SEQ ID NO:9), with the first amino acid of thisconsensus sequence present in the 27877 sequence being a serine ratherthan a glycine. The predicted active serine nucleophile is located atamino acid position 539 of both the short form and the long form of thehuman 27877 protein. Higgs et al. describe a bovine phosphatidicacid-preferring phospholipase A1 (PA-PLA1) which comprises an identicalversion of the consensus sequence (i.e., Ser-His-Ser-Leu-Gly, SEQ IDNO:9) wherein the active serine nucleophile is located at amino acidposition 540 (1998, J. Biol. Chem. 273:5468-5477). These characteristicsindicate that human 27877 protein is an active phospholipase molecule.

In another embodiment, a 27877 protein includes at least onetransmembrane domain. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 5 amino acid residues in lengththat spans the plasma membrane. More preferably, a transmembrane domainincludes about at least 10, 15, or 17 amino acid residues and spans amembrane. Transmembrane domains are rich in hydrophobic residues, andtypically have an alpha-helical structure. In a preferred embodiment, atleast 50%, 60%, 70%, 80%, 90%, or 95% or more of the amino acids of atransmembrane domain are hydrophobic, e.g., leucines, isoleucines,tyrosines, or tryptophans. Transmembrane domains are described in, forexample, Zagotta W. N. et al. (1996, Annu. Rev. Neurosci. 19: 235-263),the contents of which are incorporated herein by reference. Amino acidresidues 1 to about 620-636 of SEQ ID NOs:2 and 5 comprise atransmembrane domain in a 27877 protein.

The 27877 molecules of the present invention can further include one ormore of the N-glycosylation, cAMP-/cGMP-dependent protein kinasephosphorylation, protein kinase C phosphorylation, casein kinase IIphosphorylation, tyrosine kinase, and N-myristoylation sites describedherein, and preferably comprises most or all of them.

In an alignment of the nucleotide sequences of cDNAs encoding the longform of the human 27877 protein and the bovine PA-PLA1 (SEQ IDNO:7)(made using the ALIGN software, using default parameters, includinggap opening penalty=12, and gap extension penalty=2), the nucleic acidsequences of the cDNAs are 87.6% identical. An alignment (made using theALIGN software, using default parameters, including gap openingpenalty=12, and gap extension penalty=2) of the amino acid sequences ofthe long form of the 27877 protein and the bovine PA-(SEQ ID NO:8), theamino acid sequences of the proteins are 88.5% identical. The similarityof the sequences of human 27877 protein and bovine PA-PLA1 indicatesthat the two proteins exhibit common activities.

A hydropathy plot of the short and long forms of human 27877 wasperformed. Polypeptides of the invention include fragments whichinclude: all or part of a hydrophobic sequence e.g., the sequence ofabout residues 620-636 of either of SEQ ID NOs:2 and 5; all or part of ahydrophilic sequence, e.g., the sequence of residues 200-215 of eitherof SEQ ID NOs:2 and 5; a sequence which includes a cysteine residue; ora glycosylation site.

Because the 27877 polypeptides of the invention can modulate27877-mediated activities, they can be used to develop novel diagnosticand therapeutic agents for 27877-mediated or related disorders, asdescribed below.

As used herein, a “27877 activity,” “biological activity of 27877,” or“functional activity of 27877,” refers to an activity exerted by a 27877protein, polypeptide or nucleic acid molecule on, for example, a27877-responsive cell or on a 27877 substrate (e.g., a proteinsubstrate) as determined in vivo or in vitro. In one embodiment, a 27877activity is a direct activity, such as association with a 27877 targetmolecule. A “target molecule” or “binding partner” of a 27877 protein isa molecule with which the 27877 protein binds or interacts in nature. Inan exemplary embodiment, such a target molecule is a 27877 receptor. A27877 activity can also be an indirect activity, such as a cellularsignaling activity mediated by interaction of the 27877 protein with a27877 receptor.

The 27877 molecules of the present invention are predicted to havesimilar biological activities as phospholipase family members. Forexample, the 27877 proteins of the present invention can have one ormore of the following activities: catalyzing hydrolysis of an acyl orphosphoacyl bond of a phospholipid; catalyzing production of arachidonicacid; modulating generation of a prostaglandin; modulating generation ofa lipoxygenase reaction product formed from arachidonic acid; modulatingtumor cell invasion or metastasis; modulating tumorigenesis; modulatinga response to infection; modulating inflammation; modulating a cellularresponse to inflammation; modulating pain impulse generation; modulatingpain impulse transmission; modulating pain sensation modulatingapoptosis; modulating growth of erythroid lineage precursor cells;modulating differentiation of erythroid lineage precursor cells; andmodulating production of erythrocytes. Thus, 27877 molecules describedherein can act as novel diagnostic targets and therapeutic agents forprognosticating, diagnosing, preventing, inhibiting, alleviating, orcuring phospholipase-, lipoxygenase-, and cyclooxygenase-relateddisorders.

Other activities, as described below, include the ability to modulatefunction, survival, morphology, proliferation and/or differentiation ofcells of tissues in which 27877 molecules are expressed. Thus, the 27877molecules can act as novel diagnostic targets and therapeutic agents forcontrolling disorders involving aberrant activities of these cells.27877 molecules can also act as novel diagnostic targets and therapeuticagents for controlling cellular proliferative and/or differentiativedisorders (e.g., hematopoietic neoplastic disorders, carcinoma, sarcoma,metastatic disorders or hematopoietic neoplastic disorders, e.g.,leukemias. A metastatic tumor can arise from a multitude of primarytumor types, including but not limited to those of prostate, colon,lung, breast, and liver origin.

Based on data generated by reverse-transcriptase PCR sequence detection(i.e., “TAQMAN®”) techniques using a panel of tissues obtained fromselected human tissues, a high level of 27877 expression was observed innormal brain cortex. Brain cortex is the tissue location of numerousphysiological activities, in each of which 27877 proteins can have arole. By way of example, interconnections that are formed in normalbrain cortex tissue can mediate muscular and intellectual learning, andcan facilitate ‘neuronal learning’ processes whereby physical stimulican more easily evoke a sensation of pain over time. Because 27877protein is involved in cell signaling and differentiation processes,27877 molecules can be used to modulate muscular, intellectual, andneuronal learning processes in humans.

Various neurodegenerative disorders are known to involve aberrantfunctioning of cell signaling mechanisms. For example, Alzheimer'sdisease is often characterized by generation and accumulation ofneurofibrillary tangles containing large quantities of abnormallyphosphorylated tau protein. Although the intracellular signalingmechanisms that affect tau phosphorylation have not been fullyelucidated, it is known that numerous signaling proteins (includingthose which affect intracellular phosphate pools, such as kinase,phosphatases, and phospholipases) can affect the phosphorylation stateof tau protein. 27877 protein can influence the phosphorylation state oftau protein, and molecules that affect expression, activity, or cellularlevel of 27877 protein can be used to modulate phosphate-mediatedcellular signaling. Thus, these molecules can be used to treat patientsafflicted with neurodegenerative disorders such as Alzheimer's disease.

Because 27877 protein is involved in cell differentiation processes andis expressed in normal brain cortex, modulating expression, activity, orcellular level of 27877 protein can improve recovery of brain tissuefollowing ischemic traumas, such as those associated with stroke andtraumatic brain injuries.

Expression of 27877 was also detected in normal brain hypothalamustissue, astrocytes, and ovary tissue. Each of these cell or tissue typesis characterized by growth or proliferation during at least certainportions of the adult lifespan. Involvement of 27877 protein in celldifferentiation processes indicates that 27877 molecules can influencegrowth and proliferation of cells in these tissues, such asdifferentiation of precursor cells (e.g., neuronal stem cells orpartially differentiated brain or ovarian cell types) in a process thatleads to formation of the fully-differentiated tissue.

TAQMAN® analysis performed on a panel of selected human hematologicaltissues indicated high levels of 27877 expression in fetal liver, bonemarrow erythrocytes, in vitro generated erythroid lineage progenitorcells at day 6 through day 12, in vitro generated burst formingunit-erythroid (BFU-E), and BFU-E exposed to erythropoietin for 3 days.Each of these tissues and cell types is involved in differentiation ofblood cells and blood cell precursors. 27877 was most highly expressedin cells of the myeloid line, particularly in differentiating erythroidlineage progenitor cells (including burst-forming units of erythroidlineage; “BFU-E”) and bone marrow erythrocytes (i.e., GPA+ cells of bonemarrow). BFU-E that were exposed to erythropoietin for 3 days expressed27877 at an even greater level. Expression of 27877 was also observed inbone marrow tissue (including erythroid progenitor cells which expressedGPA at a low level), mobilized peripheral blood, and cord blood CD34+cells.

These expression data indicate that 27877 has a role in differentiationof erythroid cells, such as in terminal differentiation of these cellsto form erythrocytes. An increase in intracellular 27877 activity canlead to increased intracellular production of phosphatidic acid (PA). PAaccumulation leads to cessation of cell growth, and this may be amechanism by which the activity of 27877 protein can modulate the rateor extent of erythroid cell differentiation. PA is also known to act asan intracellular second messenger, and can regulate growth anddifferentiation of cells in which it is expressed. Involvement of 27877protein in regulation of intracellular PA indicates that 27877 moleculescan be used to modulate erythroid cell growth and differentiation, bothin normal tissues and tissues affected by a disorder, particularlydisorders which affect erythroid cell growth or differentiation.

27877 molecules can be used to enhance growth and differentiation oferythroid precursor cells to induce formation of erythrocytes andalleviate the severity of the anemia. Naturally, 27877 molecules canalso be used to inhibit or prevent anemia in situations in which anemiais anticipated (e.g., in advance of administering a hemolyticmedication).

27877 molecules can be used in non-diseased humans in order to increaseerythrocyte production. Enhanced erythrocyte production can bebeneficial, for example, during or prior to periods of intense physicalexertion or exposure to a relatively oxygen-poor altitude (e.g., at highaltitudes). These molecules can also be used to enhance oxygenabsorption by humans whose oxygen intake may be hampered byenvironmental contaminants (e.g., carbon monoxide or tobacco smoke) orby physical infirmity (e.g., diaphragm weakness associated withtraumatic injury or pneumonia).

Other disorders associated with aberrant growth and proliferation oferythrocytes include polycythemias, which are associated with anoverabundance of erythrocytes or hemoglobin. Examples of polycythemiasthat can be treated using 27877 molecules include polycythemia vera,secondary polycythemia, and relative polycythemia.

Gene Expression of 27877

Expression of 27877 was assessed in a variety of cell and tissue typesusing a standard TAQMAN™ PCR-based sequence detection procedure. Thedata collected from these experiments are summarized in Tables 2-6.

TABLE 2 Relative Tissue or Cell Type Expression Normal Artery 0.2 NormalVein 0.8 Early Aortic Smooth Muscle Cells 7.2 Coronary Smooth MuscleCells 14.3 Static Human Umbilical Vascular Epithelial Cells 8.3 ShearHuman Umbilical Vascular Epithelial Cells 8.4 Normal Heart 2.2 HeartCongestive Heart Failure 3.4 Kidney 6.4 Skeletal Muscle 1.7 NormalAdipose 0.4 Pancreas 3.2 Primary Osteoblasts 1.4 DifferentiatedOsteoclasts 0.2 Normal Skin 3.2 Normal Spinal cord 4.9 Normal BrainCortex 128 Normal Brain Hypothalamus 32.0 Nerve 3.9 Dorsal Root Ganglion9.3 Glial Cells (Astrocytes) 31.9 Glioblastoma 2.5 Normal Breast 1.2Breast Tumor 5.3 Normal Ovary 21.9 Ovary Tumor 2.9 Normal Prostate 2.0Prostate Tumor 2.7 Prostate Epithelial Cells 6.8 Normal Colon 0.6 ColonTumor 3.8 Normal Lung 0.7 Lung Tumor 5.2 Chronic Obstructive PulmonaryDisease Lung 1.7 Inflammatory Bowel Disease Colon 0.8 Normal Liver 1.4Liver fibrosis 4.3 Dermal Cells-fibroblasts 4.3 Normal Spleen 2.7 NormalTonsil 3.9 Lymph node 9.5 Resting Peripheral Blood Mononuclear Cells 1.0Skin-Decubitus 5.9 Synovium 1.8 Bone marrow mononuclear cells 2.8Activated Peripheral Blood Mononuclear Cells 3.0

TABLE 3 Relative Tissue or Cell Type Expression Lung MPI 188 1 Kidney 9Spleen 3 Fetal Liver MPI BMW54 17 Grans #9 3 NHDF Mock 1 NHLF Mock 4NHLF TGF 5 NC Heps 2 Pass Stell 2 Liver LF NDR 200 0 Liver LF NDR 191 1Lymph Nodes 11 Tonsils 3 TH0 046 6 hr 3 TH1 046 6 hr 5 TH2 046 6 hr 3CD8 6 CD14 0 CD19 2 CD3 Resting 3 MBM MNC LP139 1 mPB CD34+ LP152 1 BoneMarrow CD34+ LP154 1 Cord Blood CD34+ LP121 2 Erythroid 32 Meg 3 Neutd14 1 Bone Marrow CD15+ CD14− LP32 0 mBM CD15+ CD11b− 0 Bone Marrow GPA+14 K562 7 HL60 6 Molt4 22 Normal Hep3b 3 Hep3b Hyp 8

TABLE 4 Relative Tissue or Cell Type Expression Brain 8.7 Brain Cortex18.2 Breast 0.7 Colon Tumor 0.5 Heart 2.0 Kidney 1.3 Normal Liver 0.4Liver fib 0.5 Lung Tumor 1.2 Ovary 2.4 mBM CD34+ LP92 1.3 mBM CD34+LP143 1.2 mPB CD34+ LF70 1.2 mPB CD34+ LF162 1.1 Bone Marrow CD34+ LF930.4 Bone Marrow CD34+ LP154 2.0 Cord Blood CD34+ LP121 1.7 Cord BloodCD34+ LF101 2.1 GPA+ High LP34-1 9.8 GPA+ High 9.5 GPA+ High 69 5.6 GPA+High 74 3.2 GPA+ Low LP69 5.4 GPA+ Low LP82 3.6 In Vitro Erythroid 24hours LF102 3.9 In Vitro Erythroid 48 hours LF87 2.7 In Vitro Erythroid48 hours LF102 4.0 In Vitro Erythroid 48 hours LF72 7.6 In VitroErythroid day 6 LP31-1 7.8 In Vitro Erythroid day 6 LF113 21.1 In VitroErythroid day 7 LF24-5 13.6 In Vitro Erythroid day 8 LF113 24.2 In VitroErythroid day 10 LP24-4 16.0 In Vitro Erythroid day 12 LF23-8 13.1 InVitro Erythroid day 12 LF24-10 13.0 In Vitro Erythroid day 12 LF113 13.5In Vitro Erythroid day 14 GPA+ LP31-4 3.7 In Vitro Burst-Forming Unitday 7 LP79 10.6 In Vitro Burst-Forming Unit day 7 LP95 10.9 In VitroBurst-Forming Unit day 7 + 3 days 10.9 Erythropoietin LP81 In VitroBurst-Forming Unit day 7 + 3 days 15.6 Erythropoietin LP104

TABLE 5 Relative Tissue or Cell Type Expression Lung 2 Colon MPI 60 2Spleen MPI 380 4 Kidney MPI 58 2 Liver NDR 200 0 Fetal Liver MPI 133 31Sk Muscle MPI 167 2 mBM MNC LP140 2 mBM MNC LP7 0 mBM CD34+ LP92 4 mBMCD34+LP143 2 mPB CD34+ LF70 1 mPB CD34+ LP162 1 Bone Marrow CD34+ LF93 1Bone Marrow CD34+ LP154 2 Cord Blood CD34+ LP163 3 Cord Blood CD34+LP101 2 Bone Marrow GPA+ LF74 4 Bone Marrow GPA+ LP34-1 18 Bone MarrowGPA^(Low) LP69 5 Bone Marrow GPA^(Low) LP82 6 mPB CD41+ CD14− LP119 0Bone Marrow CD41+ CD14− LP132 1 mBMCD15+ LP15 0 mBM CD15+ CD11b− LF120 0mBM CD15+ CD11b+ LP15-2 0 Bone Marrow CD15+ CD11b− LF80-4 1 Bone MarrowCD15+ CD11b− LP23-2 0 Bone Marrow CD15+ CD11b− LF128 1 Bone Marrow CD15+CD11b+ LF128 0 Bone Marrow CD15+ CD34− LP27-2 0 Bone Marrow CD15+ CD34−LP41-1 0

TABLE 6 Relative Tissue or Cell Type Expression In Vitro Erythroid 24hours LF102 2 In Vitro Erythroid 48 hours LF73 4 In Vitro Erythroid 48hours LF87 2 In Vitro Erythroid 48 hours LF90 2 In Vitro Erythroid 48hours LF102 3 In Vitro Erythroid day 6 LP31-1 7 In Vitro Erythroid day 6LF113 16 In Vitro Erythroid day 7 LF24-5 10 In Vitro Erythroid day 8LF113 20 In Vitro Erythroid day 10 LP24-4 9 In Vitro Erythroid day 12LF23-8 13 In Vitro Erythroid day 12 LF24-10 15 In Vitro Erythroid day 12LF113 9 In Vitro Erythroid day 14 GPA+ LP31-4 2 Megs 24 hr LF102 3 Megs48 hr LF102 3 Meg d6 LF110 2 Meg d7 LP31-2 2 Meg d10 LF110 2 Meg d12LF102 1 Meg d12 LF35 1 Meg d14 LP31-5 2 Neutrophils d4 LF71 2Neutrophils d4 LF78 1 Neutrophils d6 LF26 1 Neutrophils d6 LP71 1Neutrophils d6 LP78 2 Neutrophils d7 LP41-3 1 Neutrophils d8 LF78 1Neutrophils d11 LF78 3 Neutrophils d12 LP26B 1 Neutrophils d13 LF78 3Neutrophils d14 LF71 1 Neutrophils d14 LF78 1 Neutrophils d14 LP31-6 0Platelets 0 Mast cells LP71 3 Mast Cells LP118 4 Burst-Forming Units day7 LP79 12 Burst-Forming Units day 7 LP95 12 Burst-Forming Units day 7 +3 days 15 Erythropoietin LP81 Burst-Forming Units day 7 + 3 days 18Erythropoietin LP104Human 18080

Nucleotide and corresponding amino acid sequences for a serinecarboxypeptidase family member, referred to herein as “18080” aredisclosed. 18080 protein is identical in sequence to Genbank AccessionNo. AF282618_(—)1. Applicants have shown expression of 18080 mRNA inhuman hematopoietic cells, e.g., erythroid cells, as well as, adrenalglands and endothelial cells. Accordingly, modulators of 18080polypeptide activity or expression may be used to treat or preventhematopoietic and angiogenic disorders.

The human 18080 sequence (SEQ ID NO:11), which is approximately 1921nucleotides long including untranslated regions, contains a predictedmethionine-initiated coding sequence of about 1356 nucleotides(nucleotides 33-1388 of SEQ ID NO:11; 1-1356 of SEQ ID NO:13), notincluding the terminal codon. The coding sequence encodes a 452 aminoacid protein (SEQ ID NO:12).

The human 18080 protein of SEQ ID NO:12 includes an amino-terminalhydrophobic amino acid sequence, consistent with a signal sequence, ofabout 27 amino acids (from amino acid 1 to about amino acid 27 of SEQ IDNO:12), which upon cleavage results in the production of a matureprotein form. This mature protein form is approximately 425 amino acidresidues in length (from about amino acid 28 to amino acid 452 of SEQ IDNO:12).

The serine carboxypeptidase domain of human 18080 was aligned with aconsensus amino acid sequence derived from a hidden Markov model (HMM)from PFAM. The algorithm identified two local alignments between theconsensus amino acid sequence and human 18080. Two consensus amino acidsequences (SEQ ID NOs:14 and 15), aligned to amino acids 42 to 236 and337 to 451 of SEQ ID NO:12.

Human 18080 contains the following regions or other structural features:a serine carboxypeptidase domain (PROSITE Accession Number PDOC00122)including a serine carboxypeptidase-serine active site (PS00131) and aserine carboxypeptidase-histidine active site (PS00560) located at aboutamino acid residues 42 to 236 and 337 to 451 of SEQ ID NO:12,respectively; three predicted N-glycosylation sites (PS00001) located atabout amino acids 64 to 67, 126 to 129, and 362 to 365 of SEQ ID NO:12;one predicted cAMP/cGMP-dependent protein kinase phosphorylation sites(PS00004) located at about amino acids 101 to 104 of SEQ ID NO:12; fourpredicted Protein Kinase C phosphorylation sites (PS00005) located atabout amino acids 44 to 46, 61 to 63, 188 to 190, and 417 to 419 of SEQID NO:12; six predicted Casein Kinase II phosphorylation sites (PS00006)located at about amino acids 204 to 207, 220 to 223, 280 to 283, 284 to287, 351 to 354, and 449 to 452 of SEQ ID NO:12; and eight predictedN-myristylation sites (PS00008) located at about amino acids 22 to 27,76 to 81, 119 to 124, 169 to 174, 187 to 192, 195 to 200, 331 to 336,and 360 to 365 of SEQ ID NO:12.

For general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420.

Proteolytic enzymes that exploit serine in their catalytic activity areubiquitous, being found in viruses, bacteria, and eukaryotes. See, e.g.,Rawlings & Barrett (1994) Methods Enzymol. 244: 19-61. Over 20 familiesof serine peptidase have been identified, these being grouped into 6clans on the basis of structural similarity and other functionalevidence. Structures are known for 4 of the clans: these appear to betotally unrelated, suggesting at least four evolutionary origins ofserine peptidases. Their different evolutionary origins notwithstanding,there are similarities in the reaction mechanisms of several peptidases.Carboxypeptidase C family, like chymotrypsin and subtilisin, has acatalytic triad of serine (S), aspartate (D), and histidine (H): serineacts as a nucleophile, aspartate as an electrophile, and histidine as abase. See, e.g., Rawlings & Barrett (1994) Biochem. J. 290: 205-218.

Carboxypeptidase C includes a number of serine carboxypeptidases, whichare unusual in that their optimum activity occurs in acidic conditions.In higher organisms, serine carboxypeptidases are glycoproteins (forreviews, see Breddam (1986) Carlsberg Res. Commun. 51: 83-128), andhydrolyze COOH-terminal peptide bonds. In humans, a highly specificserine carboxypeptidase cleaves the COOH-terminal residue of angiotensinII and III, and may be involved in the regulation of blood pressure.See, e.g., Odya et al. (1978) J. Biol. Chem. 253: 5927-5931 and Odya &Erdos (1981) Methods Enzymol. 80: 460-466. In addition, serinecarboxypeptidases may be involved in degrading growth factors orextracellular matrix.

The 18080 protein contains a significant number of structuralcharacteristics in common with members of the serine carboxypeptidasefamily. Serine carboxypeptidase family members are characterized by acommon catalytic mechanism which is provided by a charge relay systeminvolving an aspartic acid residue hydrogen-bonded to a histidine, whichis itself hydrogen-bonded to a serine. The catalytic triad catalyzes ahydrolysis reaction involving a COOH-terminal peptide bond. A serinecarboxypeptidase family of proteins has two signature motifs—one motifincludes the residues of [LIVM]-x-[GTA]-E-S-Y-[AG]-[GS] (SEQ ID NO:16),which contains the catalytic serine (S); and one motif spans the regionencoded by[LIVF]-x(2)-[LIVSTA]-x-[IVPST]-x-[GSDNQL]-[SAGV]-[SG]-H-x-[IVAQ]-P-x(3)-[PSA](SEQ ID NO:17), which contains the catalytic histidine (H). Residuescorresponding to catalytic serine and histidine residues are located atamino acids 167 and 431 of SEQ ID NO:12, respectively. The amino acidsequence of 18080 is identical to the sequence of a serinecarboxypeptidase 1 precursor protein having Genbank's accession numberAAG16692 (AF282618).

As used herein, the term “serine carboxypeptidase domain” includes anamino acid sequence of about 50 to 500 amino acid residues in length,more preferably about 70 to 400 amino acid residues, or about 100 to 350amino acids and has a bit score for the alignment of the sequence to theserine carboxypeptidase domain (HMM) of at least 70 or greater.Preferably, the domain includes one motif: IFSESYGG (SEQ ID NO:18)located at about amino acids 163 to 170 of SEQ ID NO:12, which includesthe catalytic serine (located at amino acid 167 of SEQ ID NO:12), andanother motif: LAFYWILKAGHMVP (SEQ ID NO:19) located at about aminoacids 421 to 434 of SEQ ID NO:12, which includes the catalytic histidine(located at amino acid 431 of SEQ ID NO:12). The serine carboxypeptidasemotif including a catalytic serine (HMM) has been assigned the PFAMAccession Number PF00131, and the serine carboxypeptidase motifincluding a catalytic histidine (HMM) has been assigned the PFAMAccession Number PF00560. The serine carboxypeptidase domain (aminoacids 42 to 236 and 337 to 451 of SEQ ID NO:12) of human 18080 alignswith a consensus amino acid sequence derived from a hidden Markov modelderived from PFAM.

In a preferred embodiment 18080 polypeptide or protein has a “serinecarboxypeptidase domain” or a region which includes at least about 50 to500 more preferably about 70 to 400, or 100 to 350 amino acid residuesand has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homologywith a “serine carboxypeptidase domain,” e.g., the serinecarboxypeptidase domain of human 18080 (e.g., residues 42 to 236 and 337to 451 of SEQ ID NO:12).

In a hydropathy plot of human 18080, polypeptides of the inventioninclude fragments which include: all or part of a hydrophobic sequence,e.g., the sequence from about amino acid 135 to 152 of SEQ ID NO:12; andall or part of a hydrophilic sequence, e.g., the sequence of from aboutamino acid 93 to 108 of SEQ ID NO:12.

To identify the presence of a “serine carboxypeptidase” domain in a18080 protein sequence, and make the determination that a polypeptide orprotein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against the Pfam database of HMMs (e.g., thePfam database, release 2.1) using the default parameters. For example,the hmmsf program, which is available as part of the HMMER package ofsearch programs, is a family specific default program for MILPAT0063 anda score of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28(3): 405-420 and a detaileddescription of HMMs can be found, for example, in Gribskov et al. (1990)Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad.Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531;and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of whichare incorporated herein by reference. A search was performed against theHMM database resulting in the identification of two “serinecarboxypeptidase” domains in the amino acid sequence of human 18080 atabout residues 42 to 236 and 337 to 451 of SEQ ID NO:12.

A 18080 protein can further include a signal peptide, and is predictedto be a secreted protein. As used herein, a “signal peptide” or “signalsequence” refers to a peptide of about 10 to 40, preferably about 15 to30, more preferably, about 27 amino acid residues in length which occursat the N-terminus of secretory and integral membrane proteins and whichcontains a majority of hydrophobic amino acid residues. For example, asignal sequence contains at least about 10 to 40, preferably about 15 to30, more preferably, 27 amino acid residues, and has at least about40-70%, preferably about 50-65%, and more preferably about 55-60%hydrophobic amino acid residues (e.g., alanine, valine, leucine,isoleucine, phenylalanine, tyrosine, tryptophan, or proline). Such a“signal sequence”, also referred to in the art as a “signal peptide,”serves to direct a protein containing such a sequence to a lipidbilayer. For example, in one embodiment, a 18080 protein contains asignal sequence of about amino acids 1 to 27 of SEQ ID NO:12. The“signal sequence” is cleaved during processing of the mature protein.The mature 18080 protein corresponds to amino acids 28 to 452 of SEQ IDNO:12.

A 18080 family member can include a “serine carboxypeptidase domain” orregions homologous with a “serine carboxypeptidase domain.” A 18080polypeptide can optionally further include at least one, two, preferablythree N-glycosylation sites (PS0001); at least one cAMP- andcGMP-dependent protein kinase phosphorylation site (PS0004); at leastone, two, three, preferably four protein kinase C phosphorylation sites(PS0005); at least one, two, three, four, five, preferably six caseinkinase II phosphorylation sites (PS0006); and at least one, two, three,four, five, six, seven, preferably eight N-myristylation sites (PS0008).

Based on the above-described sequence similarities and the tissuedistribution described below, the 18080 molecules of the presentinvention are predicted to have similar biological activities as serinecarboxypeptidase family members. Thus, in accordance with the invention,a 18080 serine carboxypeptidase or subsequence or variant polypeptidemay have one or more domains and, therefore, one or more activities orfunctions characteristic of a serine carboxypeptidase family member,including, but not limited to: (1) modulating removal of COOH-terminalresidues, i.e., having carboxypeptidase activity; (2) modulating thetransfer of an acyl group from a donor to an acceptor molecule, i.e.,having acyltransferase activity; (3) modulating (e.g., stimulating) celldifferentiation, e.g., differentiation of hematopoietic cells (e.g.,differentiation of blood cells (e.g., erythroid progenitor cells, suchas CD34+ erythroid progenitors)); (4) modulating hematopoiesis, e.g.,erythropoiesis; (5) modulating cell proliferation, e.g., proliferationof hematopoietic cells (e.g., erythroid progenitor cells); (6)modulating apoptosis, of a cell, e.g., increase apoptosis of a cancercell, e.g., a leukemic cell, (e.g., an erythroleukemia cell); orsuppress apoptosis of a blood or erythroid cell; or (7) modulatingerythroid progenitors by allowing greater interaction with growthfactors or extracellular matrix.

As the 18080 polypeptides of the invention may modulate 18080-mediatedactivities, they may be useful as of for developing novel diagnostic andtherapeutic agents for 18080-mediated or related disorders, as describedbelow.

As used herein, a “18080 activity”, “biological activity of 18080” or“functional activity of 18080”, refers to an activity exerted by a 18080protein, polypeptide or nucleic acid molecule. For example, a 18080activity can be an activity exerted by 18080 in a physiological milieuon, e.g., a 18080-responsive cell or on a 18080 substrate, e.g., aprotein substrate. A 18080 activity can be determined in vivo or invitro. In one embodiment, a 18080 activity is a direct activity, such asan association with a 32229 target molecule. A “target molecule” or“binding partner” is a molecule with which a 18080 protein binds orinteracts in nature. In an exemplary embodiment, 18080 is an enzyme forpeptide or protein substrate. In other embodiments, an 18080 hasacyltransferase activity.

18080 mRNA is found primarily in hematopoietic progenitor cells (Tables7-11). High levels of 18080 mRNA expression were observed in erythroidcells. Its expression is further enhanced in the erythroid lineage andincreases as bone marrow/blood cell differentiation proceeds. Highlevels of 18080 mRNA expression were also detected in adrenal glands andhuman umbilical vein endothelial cells (HUVECS). Tables 8-11 show TaqManassays on mRNA most derived from human hematological samples, e.g., bonemarrow (BM), erythroid cells (Eryth), megakaryocytes (Meg), neutrophils(Neut), or a negative reference sample (NTC). In Table 8, 18080 mRNA washighly expressed in BM glycophorin A (GPA) positive cells, followed bymBM CD34+ cells and Eryth cells. In Table 9, 18080 mRNA expression wasobserved in GPA Hi/Lo LF 156 Eryth cells, GPA Hi/Lo LF154 Eryth cells,and Kidney cells. In Table 10, mRNA expression was detected at theindicated times in culture (e.g., 24 hrs, 48 hrs, 6 days (D6), 12 days(D12). High levels of 18080 expressions were observed in one sample ofEryth LF127 cells and in one sample of Eryth LF139 cells, especially day6 and day 12, respectively. In Table 11, mRNA expression was detected atthe indicated times in culture (e.g., 24 hrs., 48 hrs., days inculture). Moderate level of 18080 mRNA expressions were observed in MegLF 157 cells and Neut LF141 cells, especially day 10 and day 6,respecitively.

Significant expression of 18080 mRNA is found in the bone marrow, cordblood, fetal liver, and in particular, in glycophorin A-expressing (GPA)cells or CD34-expressing populations of cells from those tissues, suchas mobilized peripheral blood GPA+/CD34+ cells, normal adult bone marrowGPA+/CD34+ cells, cord blood GPA+/CD34+ cells, normal adult bone marrowGPA+/CD34+ cells, and fetal liver GPA+/CD34+ cells; as well as erythroidprogenitor cells, e.g., erythropoietin treated erythroid burst formingunits (BFUs), erythrocytes, in vitro generated erythroblasts, andmegakaryocytes (Tables 7-8). Thus, diagnostic and therapeutic methodsusing the molecules of the invention (or agents that modulate theactivity or expression of the 18080 molecules) to treat/diagnosedisorders involving the cells/tissues expressing 18080 molecules arecontemplated by the present invention. Inhibition of the 18080 moleculeswill likely stimulate erythroid progenitors by allowing greaterinteraction with growth factors or extracellular matrix.

As used herein, a “glycophorin A-positive cell” or a “glycophorinA-expressing cell—cell” refers to a cell that expresses detectablelevels of the glycophorin A antigen, preferably human glycophorin Aantigen. Glycophorin A is a 31 kD erythrocyte membrane glycoprotein, andis typically present on immature hematopoietic precursor cells andhematopoietic colony-forming cells in the bone marrow.

As used herein, a “CD34-positive cell” or a “CD34-expressing cell”refers to a cell that expresses detectable levels of the CD34 antigen,preferably human CD34 antigen. The sequence for human CD34 is providedin SwissProt Accession Number P28906. The CD34 antigen is typicallypresent on immature hematopoietic precursor cells and hematopoieticcolony-forming cells in the bone marrow, including unipotent (CFU-GM,BFU-E) and pluripotent progenitors (CFU-GEMM, CFU-Mix and CFU-blast).The CD34 is also expressed on stromal cell precursors. Terminaldeoxynucleotidyl transferase (TdT)-positive B- and T-lymphoid precursorsin normal bone also are CD34+. The CD34 antigen is typically present onearly myeloid cells that express the CD33 antigen, but lack the CD14 andCD15 antigens and on early erythroid cells that express the CD71 antigenand dimly express the CD45 antigen. The CD34 antigen is also found oncapillary endothelial cells and approximately 1% of human thymocytes.Normal peripheral blood lymphocytes, monocytes, granulocytes andplatelets do not express the CD34 antigen. CD34 antigen density ishighest on early hematopoietic progenitor cells and decreases as thecells mature. The antigen is undetectably on fully differentiatedhematopoietic cells. Approximately 60% of acute B-lymphoid leukemia'sand acute myeloid leukemia express the CD34 antigen. The antigen is notexpressed on chronic lymphoid leukemia (B or T lineage) or lymphomas.

As the 18080 polypeptides of the invention may modulate 18080-mediatedactivities, they may be useful as of for developing novel diagnostic andtherapeutic agents for 18080-mediated or related disorders, e.g.,hematopoeitic disorder (e.g., blood cell- (e.g., erythroid-) associateddisorders), endothelial cell disorders, cardiovascular disorders, aswell as angiogenic disorders (e.g., cancerous disorders and/or disordersinvolving aberrant tumor growth).

Agents that modulate 18080 polypeptide or nucleic acid activity orexpression can be used to treat anemias, in particular, drug-inducedanemias or anemias associated with cancer chemotherapy, chronic renalfailure, malignancies, adult and juvenile rheumatoid arthritis,disorders of hemoglobin synthesis, prematurity, and zidovudine treatmentof HIV infection. A subject receiving the treatment can be additionallytreated with a second agent, e.g., erythropoietin, to further amelioratethe condition.

As used herein, the term “erythropoietin” or “EPO” refers to aglycoprotein produced in the kidney, which is the principal hormoneresponsible for stimulating red blood cell production (erythrogenesis).EPO stimulates the division and differentiation of committed erythroidprogenitors in the bone marrow. Normal plasma erythropoietin levelsrange from 0.01 to 0.03 Units/mL, and can increase up to 100 to1,000-fold during hypoxia or anemia. Graber and Krantz, Ann. Rev. Med.29:51 (1978); Eschbach and Adamson, Kidney Intl. 28:1 (1985).Recombinant human erythropoietin (rHuEpo or epoietin alpha) iscommercially available as EPOGEN.RTM. (epoietin alpha, recombinant humanerythropoietin) (Amgen Inc., Thousand Oaks, Calif.) and as PROCRIT.RTM.(epoietin alpha, recombinant human erythropoietin) (Ortho Biotech Inc.,Raritan, N.J.).

Another example of an erythroid-associated disorder is erythrocytosis.Erythrocytosis, a disorder of red blood cell overproduction caused byexcessive and/or ectopic erythropoietin production, can be caused bycancers, e.g., a renal cell cancer, a hepatocarcinoma, and a centralnervous system cancer. Diseases associated with erythrocytosis includepolycythemias, e.g., polycythemia vera, secondary polycythemia, andrelative polycythemia.

Since 18080 mRNA is expressed in endothelial cells, molecules of theinvention can be used as therapeutic and diagnostic target to treatenodothelial cell related disorders, e.g., cardiovascular (e.g., bloodvessel or hematological disorders), and angiogenic disorders, e.g.,cancers or disorders involving tumor growth.

Aberrant expression or activity of the 18080 molecules may be involvedin neoplastic disorders. Accordingly, treatment, prevention anddiagnosis of cancer or neoplastic disorders related to hematopoieticcells and, in particular, cells of the erythroid lineage are alsoincluded in the present invention. Such neoplastic disorders areexemplified by erythroid leukemias, or leukemias of erythroid precursorcells, e.g., poorly differentiated acute leukemias such aserythroblastic leukemia and acute megakaryoblastic leukemia. Additionalexemplary myeloid disorders include, but are not limited to, acutepromyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. inOncol./Hemotol. 11:267-97). In particular, AML can include theuncontrolled proliferation of CD34+ cells such as AML subtypes M1 andM2, myeloblastic leukemias with and without maturation, and AML subtypeM6, erythroleukemia (Di Guglielmo's disease). Additional neoplasticdisorders include a myelodysplastic syndrome or preleukemic disorder,e.g., oligoblastic leukemia, smoldering leukemia. Additional cancers ofthe erythroid lineage include erythroblastosis, and other relevantdiseases of the bone marrow.

The molecules of the invention may also modulate the activity ofneoplastic, non-hematopoietic tissues. Accordingly, the 18080 moleculescan act as novel diagnostic targets and therapeutic agents forcontrolling one or more of cellular proliferative and/or differentiativedisorders. Examples of such cellular proliferative and/ordifferentiative disorders include cancer, e.g., carcinoma, sarcoma, ormetastatic disorders. A metastatic tumor can arise from a multitude ofprimary tumor types, including but not limited to those of lung,prostate, colon, breast, and liver origin.

Gene Expression of 18080

Table 7 depicts relative 18080 mRNA expression as determined by TaqManassays in a panel of human tissues, including artery normal, aortadiseased, vein normal, coronary SMC, Human Umbilical Vein EndothelialCells (HUVEC), heart, pancreas, skin, spinal cord, brain, adrenalglands, dorsal root gland (DRG), nerve, breast, ovary, colon, lung,liver, megakaryocytes, and erythroid. The highest 18080 mRNA expressionwas observed in erythroid, followed by adrenal glands and HUVEC cells.Moderate 18080 mRNA expression was observed in most of the other tissues

Tables 8-11 depicts relative 18080 mRNA expression as determined byTaqMan assays on mRNA most derived from human hematological samples,e.g., bone marrow (BM), erythroid cells (Eryth), megakaryocytes (Meg),neutrophils (Neut), or a negative reference sample (NTC). In Table 8,18080 mRNA was highly expressed in BM glycophorin A (GPA) positivecells, followed by mBM CD34+ cells and erythroid cells. In Table 9,18080 mRNA expression was observed in GPA Hi/Lo LF 156 Eryth cells, GPAHi/Lo LF154 erythroid cells, and kidney cells. In Table 10, mRNAexpression was detected at the indicated times in culture (e.g., 24 hrs,48 hrs, 6 days (D6), 12 days (D12). High levels of 18080 expressionswere observed in one sample of Eryth LF127 cells and in one sample ofEryth LF139 cells, especially day 6 and day 12, respectively. In Table11, 18080 mRNA expression was detected at the indicated times in culture(e.g., 24 hrs., 48 hrs., days in culture). Moderate levels of 18080 mRNAexpressions were observed in Meg LF 157 cells and Neut LF141 cells,especially day 10 and day 6, respectively. The relative tissuedistribution of 18080 mRNA is depicted in tabular form in Tables 8-11.

TABLE 7 Tissue Type Mean β 2 Mean ∂∂ Ct Expression Artery normal 27 21 618 Aorta diseased 29 23 6 16 Vein normal 27 20 7 10 Coronary SMC 28 21 612 HUVEC 26 21 5 33 Hemangioma 29 21 8 3 Heart normal 27 21 7 11 HeartCHF 28 21 6 12 Kidney 27 21 6 15 Skeletal Muscle 30 23 7 7 Liver normal30 20 10 1 Small intestine normal 31 21 10 1 Adipose normal 28 20 8 5Pancreas 31 23 9 3 primary osteoblasts 28 20 8 4 Bladder-Female normal28 20 8 5 Adrenal Gland normal 25 20 5 30 Pituitary Gland normal 28 21 85 Brain Cortex normal 30 23 6 13 Brain Hypothalamus normal 29 22 7 6Nerve 29 21 7 6 DRG (Dorsal Root Ganglion) 29 22 8 5 Breast normal 27 216 17 Breast tumor/IDC 29 20 8 3 Ovary normal 28 21 8 5 Ovary Tumor 27 216 11 Prostate BPH 27 20 7 11 Prostate Adenocarcinoma 28 21 7 7 Colonnormal 28 20 8 4 Colon Adenocarcinoma 30 22 8 5 Lung normal 27 19 8 4Lung tumor 28 22 6 13 Lung COPD 26 19 7 6 Colon IBD 32 21 11 1 Synovium29 20 9 2 Tonsil normal 28 19 8 3 Lymph node normal 29 21 8 3 Liverfibrosis 31 22 9 2 Spleen normal 29 19 10 1 Macrophages 25 18 7 7Progenitors (erythroid, 29 20 8 3 megakaryocyte, neutrophil)Megakaryocytes 28 20 7 6 Activated PBMC 30 17 13 0 Neutrophils 30 19 111 Erythroid 27 22 5 32 positive control 28 22 6 12

TABLE 8 18080 B2 relative exp. lung 24 18 24 kidney 23 19 53 spleen 2620 14 fetal liver 29 24 31 grans. 28 21 9 NHDF mock 24 18 22 NHDF TGF 2419 31 NHLF mock 24 19 22 NHLF TGF 24 18 23 NC Heps 27 20 6 Pass. Stel.24 18 21 liver 27 20 8 NDR200 29 22 7 NDR191 27 21 14 NDR079 25 20 30lymph node 25 18 10 tonsil 24 19 22 Th0 27 17 1 Th1 27 17 1 Th2 27 17 1CD4 29 21 3 CD8 26 18 4 CD14 22 17 32 CD19 27 20 8 CD3 27 18 2 mBM CD34+23 18 42 mPB CD34+ 27 19 3 BM CD34+ 26 20 15 Cord Blood 27 19 4 Ery. 2218 42 Megs. 27 21 19 neut. 26 19 5 CD15+ 14− 22 17 20 mBM CD15+ 11b− 2318 22 BM GPA+ 25 22 132 K562 25 20 26 HL60 23 18 36 molt 25 20 18Hep36norm 25 20 37 Hep36 Hyp 25 21 45 NTC 40 40

TABLE 9 18080 Beta Rel exp Lung CHT 330 28 22 12 Heart PT 262 29 22 9Spleen 380 27 20 5 Kidney 27 25 21 58 Liver NDR 379 30 22 3 Fetal LiverBWH 54 29 22 7 Brain MCL 400 27 21 16 Colon PIT 259 28 21 7 Muscle PIT284 30 23 7 mBM MNC LP7 25 19 13 mBM CD34+ LP92 28 20 5 mPB CD34+ LP35030 20 1 mPB CD34+ LF53 26 20 16 BM CD34+ LF89 27 19 6 BM CD34+ LF75 2619 12 Cord Blood CD34+ MF1 29 22 5 Cord Blood CD34+ LF101 27 19 3 GPA HiLF154 27 22 39 GPA Hi LF156 26 21 20 GPA Lo LF154 26 21 31 GPA Lo LF15627 21 25 MF11 Stromal D32 25 19 13 post irrad MF12 Stromal cntl 24 18 12MF13 Stromal D2 24 17 8 post irrad

TABLE 10 18080 Beta Rel exp mBM CD61+ LP196 26 21 26 Platelets LP57 3922 0 mBM CD14−/11b−/15+ LF120 25 19 21 BM CD14−/11b−/15+ LF54 25 20 25BM CD14−/11b−/15+ LF128 26 19 6 BM CD14−/11b−/15+ LF145 25 20 16 mBMCD14−/11b+/15+ LF120 26 20 11 BM CD14−/11b+/15+ LF106 25 19 23 BMCD11b+/15+ LF128 26 20 19 BM-1 CD15+ ench LP41 26 20 16 Eryth D0 LF12730 20 2 Eryth 48 hr LF127 31 24 5 Eryth D6 LF127 26 22 97 Eryth D12LF127 27 21 21 Eryth D0 LF139 31 22 1 Eryth 24 hr LF139 32 23 1 Eryth 48hr LF139 30 21 2 Eryth D6 LF139 27 22 26 Eryth D12 LF139 26 22 47 BFUEryth D7 LP79 27 21 17 BFU Eryth D7 LP95 28 21 14 BFU Eryth D7 +3epoLP81 28 21 11 BFU Eryth D7 +3epo LP104 27 22 17 Mast Cell LP118 27 21 20

TABLE 11 18080 Beta Rel exp Meg D0 LF140 28 20 4 Meg 48 hr LF140 29 20 2Meg D6 LF140 27 20 7 Meg D12 LF140 26 20 9 Meg 24 hr LF102 30 20 1 Meg48 hr LF102 29 20 2 Meg 48 hr LF110 31 21 1 Meg D9 LP127 27 20 6 Meg D10LF110 27 19 6 Meg D12 LF26 27 21 10 Meg 24 hr LF157 30 20 2 Meg 48 hrLF157 29 20 2 Meg D6 LF157 27 19 6 Meg D10 LF157 26 20 14 Meg D0 LF16629 19 1 Meg 24 hr LF166 30 19 1 Meg 48 hr LF166 30 20 1 Meg D6 LF166 2820 4 Meg D10 LF166 27 20 6 Neut D0 LF141 27 19 4 Neut 48 hr LF141 27 195 Neut D6 LF141 26 19 10 Neut D12 LF141 25 18 7 Neut D0 LF144 29 22 7Neut 48 hr LF144 26 19 11 Neut D6 LF144 28 21 5 Neut D12 LF144 28 21 8NTC 40 40Human 14081

The present invention is based, in part, on the discovery of a serineprotease, referred to herein as “14081”. The transporter molecule of theinvention shares characteristics with members of the trypsin-like familyof serine proteases.

The human 14081 sequence (SEQ ID NO:20), which is approximately 980nucleotides long including untranslated regions, contains a predictedmethionine-initiated coding sequence of about 780 nucleotides, notincluding the termination codon (nucleotides 18-797 of SEQ ID NO:20;1-780 of SEQ ID NO:22). The coding sequence encodes a 260 amino acidprotein (SEQ ID NO:21).

Human 14081 contains the following regions or other structural features(for general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420 and a trypsin-like domain located at about amino acidnucleotides 4 to 242 of SEQ ID NO:21; two transmembrane domains(predicted 106 to 122 and 203 to 219 of SEQ ID NO:21; two protein kinaseC phosphorylation sites (Prosite PS00005) located at about amino acids158 to 160, and 177 to 179 of SEQ ID NO:21; three casein kinase IIphosphorylation sites (Prosite PS00006) located at about amino acids 91to 94, 135 to 138, and 218 to 221 of SEQ ID NO:21; two N-glycosylationsites (Prosite PS00001) located at about amino acids 25 to 28 and 49 to52 of SEQ ID NO:21; and four N-myristoylation sites (Prosite PS00008)located at about amino acids 7 to 12, 26 to 31, 32 to 37, and 88 to 93of SEQ ID NO:21.

A hydropathy plot of human 14081 was performed. Polypeptides of theinvention include fragments which include: all or part of a hydrophobicsequence, e.g., the sequence from about amino acid 25 to 45 (a sequencethat includes a glycosylation site at position 24 to 28), from about 52to 62, from about 91 to 122, and from about 203 to 219 of SEQ ID NO:21;all or part of a hydrophilic sequence, e.g., the sequence from aboutamino acid 6 to 32, from about 131 to 146, from about 166 to 181, andfrom about 222 to 232 of SEQ ID NO:21.

Proteases are enzymes that cleave proteins at single, specific peptidebonds. Proteases can be classified into four generic classes; serine,thiol or cysteinyl, acid or aspartyl, and metalloproteases (Cuypers etal., J. Biol. Chem. 257:7086 (1982)). Proteases are essential to avariety of biological activities, such as digestion, formation anddissolution of blood clots, reproduction, cell growth, and the mountingof an immune reaction to foreign cells and organisms. Aberrantproteolysis is associated with a number of disease states in man andother mammals.

The serine proteases include enzymes such as elastase (human leukocyte),cathepsin G, plasmin, C-1 esterase, C-3 convertase, urokinase,plasminogen activator, acrosin, and kallikreins. The trypsin-likesubclass of serine proteases include chymotrypsin, trypsin, thrombin,plasmin, Factor Xa. Certain trypsin-like proteases such as thrombin,plasmin, and Factor Xa, occupy a central role in hemostasis andthrombosis.

Homeostasis, the control of bleeding, is regulated by the physiologicalproperties of vasoconstriction and coagulation. Under normal hemostaticcircumstances, the body maintains an acute balance between clotformation and clot removal (fibrinolysis). The blood coagulation cascadeinvolves the conversion of a variety of inactive enzymes (zymogens) intoactive enzymes which ultimately convert the soluble plasma proteinfibrinogen into an insoluble matrix of highly cross-linked fibrin,Davie, E. J. et al., “The Coagulation Cascade: Initiation, Maintenanceand Regulation,” Biochemistry, 30, 10363-10370 (1991). The coagulationcascade is initiated with the activation of Factor X by activated FactorVII and Tissue Factor. Factor Xa and Factor VIIa are both trypsin-likeserine proteases, which are involved in platelet activation and thrombusformation. In certain diseases of the cardiovascular system, deviationsfrom normal hemostasis push the balance of clot formation and clotdissolution towards life-threatening thrombus formation when thrombiocclude blood flow in coronary vessels (myocardial infarctions) or limband pulmonary veins (venous thrombosis).

Proteases are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize protease enzymes. The present inventionadvances the state of the art by providing a human serine protease. Theinvention further provides the opportunity to identify inhibitors and/oractivators of a serine proteolytic enzyme, which may be useful intreating thrombosis-related and other serine protease-related disorders.

The 14081 protein contains a number of structural characteristics incommon with members of the serine protease family. Among thesecharacteristics are domains required for substrate binding, specificity,and catalysis. In particular serine proteases have a critical serineresidue in the active site or catalytic domain of the enzyme that isrequired for catalysis. Typically, the catalytic domain has theconsensus sequence -G-D-S-G-G-P-L- (SEQ ID NO:24) surrounding the activeSer residue.

As used herein, the term “serine protease” includes a protein orpolypeptide that is capable of degrading protein, which has a serineresidue at its catalytic center. A specific class of serine proteases,the trypsin-like serine proteases, share homology with the proteasetrypsin. Some trypsin-like serine proteases (e.g., trypsin,chymotrypsin, and elastase) are digestive enzymes that catalyze thebreakdown of protein in food. Other trypsin-like serine proteases (e.g.,thrombin, plasmin, factor Xa) participate in the regulation of thecoagulation cascade to regulate homeostasis. Trypsin-like and otherserine proteases differ in their protein specificity that is, each isactive only against the peptide bonds in protein molecules that havecarboxyl groups donated by certain amino acids. For the enzyme trypsin,these amino acids are arginine and lysine, for chymotrypsin they aretyrosine, phenylalanine, tryptophan, methionine, and leucine. Trypsin isthe most discriminating of all the proteolytic enzymes in terms of therestricted number of chemical bonds that it will attack. Trypsin cleavesvery specifically at R—X and K—X bonds. If X═P, no cleavage occurs.

Members of a serine protease family of proteins share a common catalyticmechanism characterized structurally by the possession of a reactiveserine (Ser) residue that is essential for their enzymatic activity.Conserved histidine (His) (e.g., located anywhere from residues 41-46 ofSEQ ID NO:21) and arginine (Arg) residues, which with Ser (locatedanywhere from residues 193-204 of SEQ ID NO:21) make up what is known asthe catalytic triad, are also catalytically essential. The His and Serresidues are located at the substrate-binding site together with theconserved Asp, which is commonly buried in a solvent inaccessible pocketin a folded serine protease protein. Alignment among family members ofthe trypsin-like proteases (e.g., trypsin, chyrmotrypsin, bovinetrypsin, and pocine elastase) shows that these enzymes are about 40%identical in their internal sequences, and their internal sequences areeven more alike (Voet & Voet, Biochemistry, John Wiley & Sons, New York,N.Y. p. 373-382 (1990)).

A 14081 polypeptide can include a “serine protease domain” or regionshomologous with a “serine protease domain”. A 14081 polypeptide canfurther include a “trypsin-like serine protease domain” or regionshomologous with a “trypsin-like serine protease domain.” and at leastone catalytic triad.

As used herein, the term “trypsin-like domain” includes an amino acidsequence of about 4 to 242 amino acid residues in length and having abit score for the alignment of the sequence to the trypsin-like domain(HMM) of at least 280. Preferably a trypsin-like domain mediatesproteolytic degradation of proteins and polypeptides. Preferably, atrypsin-like domain includes at least about 5 to 10 amino acids, morepreferably about 10 to 100 amino acid residues, more preferably 100 to200, or about 200 to 250 amino acids and has a bit score for thealignment of the sequence to the trypsin-like domain (HMM) of at least50, more preferably 100, most preferably 200 or greater.

As mentioned above, the trypsin-like domain can include a trypsin-likecatalytic domain having a catalytic triad. In the above conservedsignature sequence, and other motifs or signature sequences describedherein, the standard IUPAC one-letter code for the amino acids is used.Each element in the pattern is separated by a dash (-); square brackets([ ]) indicate the particular residues that are accepted at thatposition; x indicates that any residue is accepted at that position; andnumbers in parentheses (( )) indicate the number of residues representedby the accompanying amino acid. The consensus sequence surrounding theactive site of trypsin is -G-D-S-G-G-P-L- (SEQ ID NO:24) located aboutamino acids 197 to 203 of SEQ ID NO:21 of human 14081 polypeptide. Thetrypsin-like domain (amino acids 4 to 242 of SEQ ID NO:21) of human14081 aligns with the trypsin consensus amino acid sequence (SEQ IDNO:23) derived from a hidden Markov model.

In a preferred embodiment, a 14081 polypeptide or protein has a“trypsin-like domain” or a region which includes at least about 5 to 10more preferably about 100 to 200 or 200 to 250 amino acid residues andhas at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a“trypsin-like domain,” e.g., the trypsin-like domain of human 14081(e.g., residues 4 to 242 of SEQ ID NO:21).

To identify the presence of a “trypsin-like” domain in a 14081 proteinsequence, and make the determination that a polypeptide or protein ofinterest has a particular profile, the amino acid sequence of theprotein can be searched against the Pfam database of HMMs (e.g., thePfam database, release 2.1) using the default parameters. For example,the hmmsf program, which is available as part of the HMMER package ofsearch programs, is a family specific default program for MILPAT0063 anda score of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28:405-420 and a detailed descriptionof HMMs can be found, for example, in Gribskov et al. (1990) Meth.Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference. A search was performed against the HMMdatabase resulting in the identification of a “trypsin-like domain”domain in the amino acid sequence of human 14081 at about residues 4 to242 of SEQ ID NO:21.

A 14081 polypeptide can include at least one, preferably two“transmembrane domains” or regions homologous with a “transmembranedomain”. As used herein, the term “transmembrane domain” includes anamino acid sequence of about 10 to 40 amino acid residues in length andspans the plasma membrane. Transmembrane domains are rich in hydrophobicresidues, e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more ofthe amino acids of a transmembrane domain are hydrophobic, e.g.,leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domainstypically have alpha-helical structures and are described in, forexample, Zagotta et al., (1996) Annual Rev. Neurosci. 19:235-263, thecontents of which are incorporated herein by reference. Thetransmembrane domains of human 14081 are located at about residues 106to 122 and about residues 203 to 219 of SEQ ID NO:21.

To identify the presence of a “transmembrane” domain in a 14081 proteinsequence, and make the determination that a polypeptide or protein ofinterest has a particular profile, the amino acid sequence of theprotein can be analyzed by a transmembrane prediction method thatpredicts the secondary structure and topology of integral membraneproteins based on the recognition of topological models (MEMSAT, Joneset al., (1994) Biochemistry 33:3038-3049).

A 14081 polypeptide can include at least one, preferably three“non-transmembrane regions.” As used herein, the term “non-transmembraneregion” includes an amino acid sequence not identified as atransmembrane domain. The non-transmembrane regions in 14081 are locatedat about amino acids 1 to 105, 123 to 202, and 220 to 260 of SEQ IDNO:21. The second non-transmembrane domain (amino acids 123 to 202) ispredicted to be intracellular.

The non-transmembrane regions of 14081 include at least one cytoplasmicregion. In one embodiment, a 14081 cytoplasmic region includes at leastone, cytoplasmic loop. As used herein, the term “loop” includes an aminoacid sequence which is not included within a phospholipid membrane,having a length of at least about 4, preferably about 5 to 30, morepreferably about 6 to 60, most preferably 6 to 80 or more amino acidresidues, and has an amino acid sequence that connects two transmembranedomains within a protein or polypeptide. Accordingly, the N-terminalamino acid of a loop is adjacent to a C-terminal amino acid of atransmembrane domain in a 14081 molecule, and the C-terminal amino acidof a loop is adjacent to an N-terminal amino acid of a transmembranedomain in a 14081 molecule. As used herein, a “cytoplasmic loop”includes a loop located inside of a cell or within the cytoplasm of acell. For example, a “cytoplasmic loop” can be found at about amino acidresidues 123 to 202 of SEQ ID NO:21.

In a preferred embodiment, a 14081 polypeptide or protein has acytoplasmic loop or a region which includes at least about 4, preferablyabout 5 to 30, and more preferably about 6 to 60, most preferably 6 to80 or more amino acid residues and has at least about 60%, 70% 80% 90%95%, 99%, or 100% homology with a cytoplasmic loop,” e.g., a cytoplasmicloop of human 14081 (e.g., residues 123 to 202 of SEQ ID NO:21).

In another embodiment, a 14081 non-transmembrane region includes atleast one, two, preferably three non-cytoplasmic loops. As used herein,a “non-cytoplasmic loop” includes a loop located outside of a cell orwithin an intracellular organelle. Non-cytoplasmic loops includeextracellular domains (i.e., outside of the cell) and intracellulardomains (i.e., within the cell). When referring to membrane-boundproteins found in intracellular organelles (e.g., mitochondria,endoplasmic reticulum, peroxisomes microsomes, vesicles, endosomes, andlysosomes), non-cytoplasmic loops include those domains of the proteinthat reside in the lumen of the organelle or the matrix or theintermembrane space. For example, a “non-cytoplasmic loop” can be foundat about amino acid residues 123 to 202 of SEQ ID NO:21.

In a preferred embodiment, a 14081 polypeptide or protein has at leastone non-cytoplasmic loop or a region which includes at least about 4,preferably about 5 to 30, more preferably about 6 to 60 most preferably6 to 80 or more amino acid residues and has at least about 60%, 70% 80%90% 95%, 99%, or 100% homology with a “non-cytoplasmic loop,” e.g., atleast one non-cytoplasmic loop of human 14081 (e.g., residues 1 to 105,123 to 202, and 220 to 260 of SEQ ID NO:21).

A human 14081 protein can further include at least one tyrosine kinasephosphorylation site (e.g., at residues 48 to 56 and 167 to 173) or anamidation site (e.g., at residues 189 to 192) or a glycosylation site(e.g., at residues 25 to 28 and 49 to 52) or a myristoylation site(e.g., at residues 7 to 12, 26 to 31, 32 to 37, and 88 to 93).

A 14081 family member can include at least one trypsin-like domains; andoptionally a transmembrane or non-transmembrane domain. Furthermore, a14081 family member can include at least one, preferably two proteinkinase C phosphorylation sites (Prosite PS00005); at least one, two, andpreferably three casein kinase II phosphorylation sites (PrositePS00006); at least one, preferably two N-glycosylation sites (PrositePS00001); and at least one, two, three, and preferably fourN-myristoylation sites (Prosite PS00008).

As the 14081 polypeptides of the invention can modulate 14081-mediatedactivities, they can be useful for developing novel diagnostic andtherapeutic agents for trypsin-like serine protease-associated or other14081-associated disorders, as described below.

As used herein, a “serine protease-associated activity” includes anactivity which involves “trypsin-like serine protease activity,” whichdegrade proteins with varying specificity. Members of this family canplay a role in diseases involving biological activities such asdigestion formation and dissolution of blood clots, reproduction, cellgrowth, and the immune reaction to foreign cells and organisms. Suchdiseases include cardiovascular and non-cardiovascular diseases such asatherosclerosis, myocardial infarction, unstable angina, stroke,restenosis, deep vein thrombosis, disseminated intravascular coagulationcaused by trauma, reperfusion damage, sepsis or tumor metastasis,hemodialysis, cardiopulmonary bypass surgery, atherectomy, arterialstent placement, adult respiratory distress syndrome, edotoxic shock,rheumatoid arthritis, ulcerative colitis, induration, metastasis,hypercoagulability during chemotherapy, adult respiratory distresssyndrome, Alzheimer's disease, Parkinson's disease, Down's syndrome,inflammation such as edema, pancreatitis, and cancer.

As used herein, a “14081 activity”, “biological activity of 14081” or“functional activity of 14081”, refers to an activity exerted by a 14081protein, polypeptide or nucleic acid molecule on e.g., a14081-responsive cell or on a 14081 substrate, e.g., a proteinsubstrate, as determined in vivo or in vitro. In one embodiment, a 14081activity is a direct activity, such as an association with a 14081target molecule. A “target molecule” or “binding partner” is a moleculewith which a 14081 protein binds or interacts in nature. In an exemplaryembodiment, 14081 is a receptor (or transporter or protease), e.g., atrypsin-like protease, and thus binds to or interacts in nature with amolecule (or protein substrate), e.g., an organic ion (or signalpeptide).

In an exemplary embodiment, 14081 is an enzyme for a protein orpolypeptide substrate.

A 14081 activity can also be an indirect activity, e.g., a cellularsignaling activity mediated by interaction of the 14081 protein with a14081 receptor. Based on the above-described sequence structures andsimilarities to molecules of known function, the 14081 molecules of thepresent invention can have similar biological activities as trypsin-likeserine protease family members. For example, the 14081 proteins of thepresent invention can have one or more of the following activities: (1)the ability to degrade proteins; and (2) the ability to phosphorylatecarbohydrates. The ability to degrade proteins is based on the abilityto bind, hydrolyze, and release a protein. The catalytic mechanism ofserine proteases has been studies extensively. In general, to bind amolecule, the serine protease binds a protein substrate to form aMichaelis complex and the Ser residue nucleophilically attacks thescisslile peptide's carbonyl group to form a tetrahedral intermediate,wherein the Asp remains a carboxylate ion. The tetrahydral intermediatehas a well defined, although transient existence. During the hydrolysisstep, the tetrahedral intermediate decomposes to an acyl-enzymeintermediate under the driving force of proton domation from the His.The amine leaving group is released from the enzyme and replace by waterfrom the solvent. The acyl-enzyme intermediate is extremely unstable togydrolytic cleavage because of the enzyme's catalytic properties. Next,a deacylation step proceeds largely through the reversal of the previoussteps with the release of the carboylate product (the new C-terminalportion of the cleaved polypeptide chain) and the concomitantregeneration of the enzyme.

The 14081 molecules of the invention can modulate the activities ofcells in tissues where they are expressed. For example, TaqMan anysisshows that 14081 mRNA is expressed in hemangioma, kidney, pituitary,spinal cord, prostate tumor, human umbilical vein endothelial cells,hypothalamus, normal breast, bone marrow megakaryocytes, isolated CD61+cells, brain cortex, tonsil, and platelets from patients with ischemicheart disease. Accordingly, the 14081 molecules of the invention can actas therapeutic or diagnostic agents for renal, hormonal, endocrine,neurological, hyperprolifereative, reproductive, breast, hematologicaland inflammatory disorders.

As a preferred embodiment, the 14081 molecules can be used to treatcoagulation-related disorders in part because the 14081 mRNA isexpressed in the platelets of patients with ischemic heart disease. Inaddition, 14081 levels are increased in samples from patients withcoronary artery disease. Thus, the 14081 molecules can act as noveldiagnostic targets and therapeutic agents for controlling one or morecoagulation or other serine protease or trypsin-like serine proteasedisorders. As used herein, “serine protease disorders” or “trypsin-likeserine protease disorders” are diseases or disorders whose pathogenesisis caused by, is related to, or is associated with aberrant or deficientserine protease or trypsin-like serine protease protein function orexpression. Examples of such disorders, e.g., trypsin-like serineprotease-associated or other 14081-associated disorders, include but arenot limited to, cellular proliferative and/or differentiative disorders,disorders associated with metabolism (e.g., hormonal), immune e.g.,inflammatory, disorders, cardiovascular disorders, endothelial celldisorders, renal disorders, neurological disorders, hyperprolifereativedisorders, reproductive disorders, breast disorders, and hematologicaldisorders.

The 14081 molecules can be used to treat cellular proliferative and/ordifferentiative disorders in part because trypsin-like serine proteasefamily members are found in the prostate tumors. Examples of cellularproliferative and/or differentiative disorders include cancer, e.g.,carcinoma, sarcoma, metastatic disorders or hematopoietic neoplasticdisorders, e.g., leukemias. A metastatic tumor can arise from amultitude of primary tumor types, including but not limited to those ofprostate, colon, lung, breast and liver origin.

The 14081 molecules can be used to treat immune disorders in partbecause trypsin-like serine protease family members are found in thebone marrow megakaryocytes, CD61+ cells, and platelets. Moreparticularly, the 14081 nucleic acid and protein of the invention can beused to treat and/or diagnose a variety of immune, e.g., inflammatory,(e.g. respiratory inflammatory) disorders, as described herein.

The 14081 molecules can be used to treat cardiovascular disorders inpart because trypsin-like serine protease family members are found inthe platelets and participate in platelet activation and thrombusformation. In addition, 14081 levels are increased in samples frompatients with coronary artery disease. 14081 may cleave and activatechannels regulating platelet function. Antagonizing 14081 will blockplatelet activation.

The 14081 molecules can be used to treat endothelial cell disorders inpart because trypsin-like serine protease family members are found inthe human umbilical endothelial cells.

The 14081 molecules can be used to treat metabolic disorders in partbecause trypsin-like serine protease family members are found in thepituitary gland. 14081 can play an important role in the regulation ofmetabolism or pain disorders.

Human 32140

The present invention is based, in part, on the discovery of a novelhuman aldehyde dehydrogenase, referred to herein as “32140”.

The human 32140 sequence (SEQ ID NO:25), which is approximately 7220nucleotides long including untranslated regions, contains a predictedmethionine-initiated coding sequence of about 2769 nucleotides(nucleotides 129-2897 of SEQ ID NO:25; nucleotides 1-2769 of SEQ IDNO:27), not including the terminal codon. The coding sequence encodes a923 amino acid protein (SEQ ID NO:26).

Human 32140 contains the following regions: a predicted aldehydedehydrogenase domain (PFAM Accession PF00171) located at about aminoacid residues 450-923 of SEQ ID NO:26, and a predicted formyltransferase (PFAM Accession PF00551) located at about amino acidresidues 23-202 of SEQ ID NO:26.

The 32140 protein also includes the following domains: a“10-formyltetrahydrofolate dehydrogenase domain” at about amino acidresidues 265-336 of SEQ ID NO:26, and a“formyltransferase/methyltransferase domain” at about amino acidresidues 211-328 of SEQ ID NO:26.

For general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420.

A plasmid containing the nucleotide sequence encoding human 32140 wasdeposited with American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110-2209, on Jun. 1, 2001 and assignedAccession Number PTA-3424. This deposit will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure. Thisdeposit was made merely as a convenience for those of skill in the artand is not an admission that a deposit is required under 35 U.S.C. §112.

The 32140 protein contains a significant number of structuralcharacteristics in common with members of the aldehyde dehydrogenasefamily. As used herein, the term “aldehyde dehydrogenase” refers to aprotein or polypeptide which is capable of catalyzing an aldehydeoxidation reaction. Aldehyde dehydrogenases can have a specificity forvarious aldehyde precursors. An aldehyde dehydrogenase polypeptidetypically includes a region of sequence similarity that comprises boththe NAD⁺/NADP binding site and the enzyme active site (Vasiliou et al.,(1999) Pharmacogenetics 9:421-434). This region of sequence similarityis located at about amino acids 669-728 of SEQ ID NO:26. The 32140polypeptide exhibits sequence identity with the aldehyde dehydrogenasefamily at four key amino acid residues that have been shown to beimportant for aldehyde dehydrogenase function, including a glutamateinvolved in catalytic activity (amino acid 694 of SEQ ID NO:26), acysteine involved in substrate binding (amino acid 728 of SEQ ID NO:26),and two glycines involved in NAD⁺ or NADP binding (amino acids 671 and676 of SEQ ID NO:26).

Typically, aldehyde dehydrogenases play a role in a wide variety ofcellular processes. For example, the metabolism of many amino acids,fatty acids, and glycerolipids, as well as ascorbate, aldarate,butanoate, pyruvate, propanoate, and 4-aminobutyric acid (GABA),involves specific oxidation reactions catalyzed by aldehydedehydrogenases. Aldehyde dehydrogenases also participate in retinoidsignaling, catalyzing the oxidation of retinal (which is required forvision) to retinoic acid (which plays an important role as a signalingmolecule in embryonic differentiation) (reviewed by Duester, inEnzymology and Molecular Biology of Carbonyl Metabolism, KeuwerAcademic/Plenum Publishers, 1989). Thus, the molecules of the presentinvention may be involved in one or more of: 1) the oxidation of analdehyde; 2) the modulation of amino acid metabolism; 3) the modulationof fatty acid or glycerophospholipid metabolism; 4) the modulation ofretinoic acid signaling; 5) the modulation of cell differentiation; 6)the modulation of vision; 7) the modulation of 4-aminobutyric acid(GABA) metabolism; 8) the modulation of the metabolism of drugs orenvironmental agents; 9) the modulation of alcohol metabolism; 10) themodulation of tumor cell growth and invasion; or 11) the modulation ofvitamin metabolism.

A 32140 polypeptide can include an “aldehyde dehydrogenase domain” orregions homologous with an “aldehyde dehydrogenase domain”. As usedherein, the term “aldehyde dehydrogenase domain” includes an amino acidsequence of about 80-300 amino acid residues in length and having a bitscore for the alignment of the sequence to the aldehyde dehydrogenasedomain (HMM) of at least 8. Preferably, an aldehyde dehydrogenase domainincludes at least about 100-250 amino acids, more preferably about130-200 amino acid residues, or about 160-200 amino acids and has a bitscore for the alignment of the sequence to the aldehyde dehydrogenasedomain (HMM) of at least 16 or greater. The aldehyde dehydrogenasedomain (HMM) has been assigned the PFAM Accession PF00171. The aldehydedehydrogenase domain (amino acids 450-923 of SEQ ID NO:26) of human32140 aligns with a consensus amino acid sequence derived from a hiddenMarkov model (SEQ ID NO:28).

In a preferred embodiment, 32140 polypeptide or protein has a “aldehydedehydrogenase domain” or a region which includes at least about 100-250more preferably about 130-200 or 160-200 amino acid residues and has atleast about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with an“aldehyde dehydrogenase domain,” e.g., the aldehyde dehydrogenase domainof human 32140 (e.g., amino acid residues 450-923 of SEQ ID NO:26).

To identify the presence of an “aldehyde dehydrogenase” domain in a32140 protein sequence, and make the determination that a polypeptide orprotein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against a database of HMMs (e.g., the Pfamdatabase, release 2.1) using the default parameters. For example, thehmmsf program, which is available as part of the HMMER package of searchprograms, is a family specific default program for MILPAT0063 and ascore of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al., (1997) Proteins 28(3):405-420 and a detaileddescription of HMMs can be found, for example, in Gribskov et al.,(1990) Meth. Enzymol. 183:146-159; Gribskov et al., (1987) Proc. Natl.Acad. Sci. USA 84:4355-4358; Krogh et al., (1994) J. Mol. Biol.235:1501-1531; and Stultz et al., (1993) Protein Sci. 2:305-314, thecontents of which are incorporated herein by reference.

As the 32140 polypeptides of the invention may modulate 32140-mediatedactivities, they may be useful for developing novel diagnostic andtherapeutic agents for 32140-mediated or related disorders, as describedbelow.

As used herein, a “32140 activity”, “biological activity of 32140” or“functional activity of 32140”, refers to an activity exerted by a 32140protein, polypeptide or nucleic acid molecule on e.g., a32140-responsive cell or on a 32140 substrate, e.g., a lipid or proteinsubstrate, as determined in vivo or in vitro. In one embodiment, a 32140activity is a direct activity, such as an association with a 32140target molecule. A “target molecule” or “binding partner” is a moleculewith which a 32140 protein binds or interacts in nature, e.g., analdehyde, which the 32140 protein oxidizes. A 32140 activity can also bean indirect activity, e.g., a cellular signaling activity mediated byinteraction of the 32140 protein with a 32140 ligand. For example, the32140 proteins of the present invention can have one or more of thefollowing activities: 1) the oxidation of an aldehyde; 2) the modulationof amino acid metabolism; 3) the modulation of fatty acid orglycerophospholipid metabolism; 4) the modulation of retionic acidsignaling; 5) the modulation of cell differentiation; 6) the modulationof vision; 7) the modulation of 4-aminobutyric acid (GABA) metabolism;8) the modulation of the metabolism of drugs or environmental agents; 9)the modulation of alcohol metabolism; 10) the modulation of tumor cellgrowth and invasion; or 11) the modulation of vitamin metabolism. 12)the ability to antagonize or inhibit, competitively ornon-competitively, any of 1-11.

Accordingly, 32140 protein may mediate various disorders, includingcellular proliferative and/or differentiative disorders, lung disorders,liver disorders, brain disorders, heart disorders, kidney disorders,breast disorders, and testis disorders.

The 32140 nucleic acid and protein of the invention can be used to treatand/or diagnose a variety of proliferative disorders.

The 32140 gene appears to have an important role in viral pathogenesis.In particular, Herpes Simples Virus (HSV) induces expression of thenovel 10-formyltetrahydrofolate DH encoded by the gene 32140,particularly in infected neurons. Viral panels have shown that 32140 isinduced in HSV-infected mouse ganglia both during active and latentphases. It is induced up to 5-fold in HSV-infected neuroblastoma(Ntera2). 32140 appears to be a 10-formyltetrahydrofolate DH isozymewhich is expressed in different tissues (e.g. neuronal tissues) comparedto the known 10-formyltetrahydrofolate DH. The 32140 aldehydedehydrogenase is therfore an important host gene for HSV infection andfinds use in the treatment of disorders resulting from Herpes SimplesVirus (HSV) and hepatitis B infection. Also, gene 32140 is inducedduring infection by HSV, but not infection with other viruses such asVZV, HBV, and HCV.

Gene Expression Analysis of 32140

TaqMan analysis shows 32140 was expressed in different cells: normal oruninfected liver; uninfected ganglia and ganglia infected with HerpesSimplex Virus; a time course of HSV (strain 17+) infection of humanNtera2 neuroblastoma cells (times are 0, 2.5, 5, and 7 hourspost-infection); same infection experiment using HSV strain KOS; a timecourse of Varicella Zoster virus (VZV) infected human MRC5 (lungfibroblast cells) (times are 0, 18 and 72 hours post-infection); andNtera2 cells infected with an adenovirus that expresses the HSV ICPOtranscription factor.

TaqMan analysis also shows 32140 was expressed in different tissues:“normal” liver, lung and kidney tissue; uninfected ganglia and gangliainfected with Herpes Simplex Virus; HSV (strain 17+) infection of humanNtera2 neuroblastoma cells (times are 0, 2.5, 5, and 7 hourspost-infection); a time course of Varicella Zoster virus (VZV) infectedhuman MRC5 (lung fibroblast cells) (times are 0, 18 and 72 hourspost-infection); Ntera2 cells infected with an adenovirus that expressesthe HSV ICPO transcription factor; hepatitis B virus (HBV) expressingHepG2.2.15 cells compared to the parental HepG2 cell control; andHBV-infected and hepatitis C virus (HCV)-infected liver samples.

Gene 32140 is highly expressed in salivary glands and testes, moremoderately expressed in brain, small intestine, stomach, spinal cord,and dorsal root ganglia, and it is expressed at lower levels in theother tissues indicated.

Human 50352

The present invention is based, in part, on the discovery of a novelubiquitin-protein ligase family member, referred to herein as “50352”.

The human 50352 sequence (SEQ ID NO:29), which is approximately 3513nucleotides long including untranslated regions, contains a predictedmethionine-initiated coding sequence of about 3066 nucleotides, notincluding the termination codon (nucleotides 82-3147 of SEQ ID NO:29;1-3066 of SEQ ID NO:31). The coding sequence encodes a 1022 amino acidprotein (SEQ ID NO:30).

Human 50352 contains the following regions or other structural features(for general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420:

One, two, three, preferably four and most preferably five or moreregulator of chromosome condensation domain(s) (PFAM Accession NumberPF00415; SEQ ID NO:32) located at about amino acid residues 43 to 92,from about residues 93 to 145, from about residues 146 to 198, fromabout residues 200 to 253 and from about residues 254 to 304 of SEQ IDNO:30; a homologous to the E6-AP carboxyl terminus domain (PFAMAccession Number PF00632; SEQ ID NO:33) located at about amino acidresidues 726 to 1015 of SEQ ID NO:30; a conjugation ubiquitin cyclinKIAA0032 binding CG9153 cyclin E domain (ProDom Accession NumberPD136613) located at about amino acids 374 to 645 of SEQ ID NO:30; aligase ubiquitin conjugation ubiquitin-protein 6.3.2 domain (ProDomAccession Number PD255820) located at about amino acids 758 to 811 ofSEQ ID NO:30; a ligase ubiquitin conjugation 6.3.2 domain (ProDomAccession Number PD002225) located at about amino acids 836 to 1013 ofSEQ ID NO:30; fourteen protein kinase C phosphorylation sites (PrositePS00005) located at about amino acids 118 to 120, 216 to 218, 224 to226, 319 to 321, 368 to 370, 443 to 445, 448 to 450, 647 to 649, 669 to671, 801 to 803, 850 to 852, 867 to 869, 951 to 953, and 975 to 977 ofSEQ ID NO:30; twenty five casein kinase II phosphorylation sites(Prosite PS00006) located at about amino acids 46 to 49, 124 to 127, 242to 245, 259 to 262, 290 to 293, 323 to 326, 339 to 342, 362 to 365, 375to 378, 385 to 388, 398 to 401, 431 to 434, 448 to 451, 459 to 462, 550to 553, 690 to 693, 729 to 732, 793 to 796, 807 to 810, 904 to 907, 915to 918, 939 to 942, 951 to 954, 973 to 976, and 995 to 998 of SEQ IDNO:30; four cAMP/cGMP-dependent protein kinase phosphorylation sites(Prosite PS00004) located at about amino acids 14 to 17, 394 to 397, 424to 427, and 445 to 448 of SEQ ID NO:30; two N-glycosylation sites(Prosite PS00001) located at about amino acids 263 to 266 and 865 to 868of SEQ ID NO:30; thirteen N-myristoylation sites (Prosite PS00008)located at about amino acids 19 to 24, 59 to 64, 180 to 185, 189 to 194,200 to 205, 206 to 211, 231 to 236, 295 to 300, 307 to 312, 354 to 359,426 to 431, 714 to 719, and 762 to 767 of SEQ ID NO:30; one tyrosinekinase phosphorylation site (Prosite PS00007) located at about aminoacids 750 to 758 of SEQ ID NO:30; one amidation site (Prosite PS00009)located at about amino acids 55 to 58 of SEQ ID NO:30; and one, two,three, preferably four and most preferably five regulator of chromosomecondensation signature 2 domain(s) (Prosite PS00626) located at aboutamino acids 28 to 38, 80 to 90, 133 to 143, 186 to 196, and 241 to 251of SEQ ID NO:30.

A hydropathy plot of human 50352 was performed. Polypeptides of theinvention include fragments which include: all or part of a hydrophobicsequence, e.g., the sequence from about amino acid 71 to 81, from about411 to 421, from about 475 to 490, from about 500 to 510, from about 532to 544, from about 615 to 631, from about 695 to 705, and from about 761to 772 of SEQ ID NO:30; all or part of a hydrophilic sequence, e.g., thesequence from about amino acid 5 to 20, from about 48 to 63, from about261 to 280, from about 312 to 325, from about 392 to 403, from about 439to 451, from about 551 to 560, from about 660 to 670, from about 749 to757, from about 851 to 861, and from about 903 to 928 of SEQ ID NO:30; asequence which includes a Cys, or a glycosylation site.

The 50352 protein contains a significant number of structuralcharacteristics in common with members of the ubiquitin-protein ligasefamily.

As used herein, the term “ubiquitin-protein ligase” includes a proteinor polypeptide which is capable of conjugating ubiquitin molecules toeither substrates or to ubiquitin moieties bound to substrates deemedfor degradation. Ubiquitin-protein ligases are responsible for the thirdand final step of ubiquitin conjugation, they accept ubiquitin from anE2 ubiquitin-conjugated enzyme in the form of a thioester and thentransfer the ubiquitin to a target protein by forming an isopeptide bondbetween glycine residues of ubiquitin and an alpha amino group of alysine residue of the substrate or of a previously conjugated ubiquitin.Ubiquitin-protein ligases are also responsible in determining thespecificity of ubiquitination. Members of the ubiquitin-protein ligasefamily of proteins share a region of similarity known as the homologousto the E6-AP carboxyl terminus domain. This domain is composed ofapproximately 350 amino acids and it has a conserved cysteine residuelocated in the last 32 to 46 amino acids which is necessary for theubiquitin thioester formation.

In one embodiment of the invention, a 50352 polypeptide includes atleast one, two, three, preferably four and most preferably five or moreregulator of chromosome condensation domain.

In another embodiment of the invention, a 50352 polypeptide includes atleast one “homologous to the E6-AP carboxyl terminus” domain.

A 50352 polypeptide can include a “homologous to the E6-AP carboxylterminus domain” or regions homologous with a “homologous to the E6-APcarboxyl terminus domain”. A 50352 polypeptide can further include one,two, three, preferably four, and most preferably five or more “regulatorof chromosome condensation domain(s)” or regions homologous with a“regulator of chromosome condensation domain”.

As used herein, the term “homologous to the E6-AP carboxyl terminusdomain” includes an amino acid sequence of about 250 to 350 amino acidresidues in length and having a bit score for the alignment of thesequence to the homologous to the E6-AP carboxyl terminus domain (HMM)of at least 110.9. Preferably a homologous to the E6-AP carboxylterminus domain mediates ubiquitin conjugation and specificity ofubiquitin conjugation to substrates or ubiquitin moieties bound tosubstrates. Preferably, a homologous to the E6-AP carboxyl terminusdomain includes at least about 200 to 400 amino acids, more preferablyabout 225 to 375 amino acid residues, or about 250 to 350 amino acidsand has a bit score for the alignment of the sequence to the homologousto the E6-AP carboxyl terminus domain (HMM) of at least 80, 90, 100, 110or greater.

In a preferred embodiment, a 50352 polypeptide or protein has a“homologous to the E6-AP carboxyl terminus domain” or a region whichincludes at least about 200 to 400 more preferably about 225 to 375 or250 to 350 amino acid residues and has at least about 60%, 70% 80% 90%95%, 99%, or 100% homology with a “homologous to the E6-AP carboxylterminus domain,” e.g., the homologous to the E6-AP carboxyl terminusdomain of human 50352 (e.g., residues 726 to 1015 of SEQ ID NO:30).

To identify the presence of a “homologous to the E6-AP carboxyl terminusdomain” in a 50352 protein sequence, and make the determination that apolypeptide or protein of interest has a particular profile, the aminoacid sequence of the protein can be searched against the Pfam databaseof HMMs (e.g., the Pfam database, release 2.1) using the defaultparameters. For example, the hmmsf program, which is available as partof the HMMER package of search programs, is a family specific defaultprogram for MILPAT0063 and a score of 15 is the default threshold scorefor determining a hit. Alternatively, the threshold score fordetermining a hit can be lowered (e.g., to 8 bits). A description of thePfam database can be found in Sonhammer et al. (1997) Proteins28:405-420 and a detailed description of HMMs can be found, for example,in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al.(1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J.Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci.2:305-314, the contents of which are incorporated herein by reference. Asearch was performed against the HMM database resulting in theidentification of a “homologous to the E6-AP carboxyl terminus domain”in the amino acid sequence of human 50352 at about residues 726 to 1015of SEQ ID NO:30.

A 50352 molecule can further include one, two, three, preferably four,and most preferably five or more “regulator of chromosome condensationdomain(s)”. As used herein, the term “regulator of chromosomecondensation domain” includes amino acid sequence(s) of about 40 to 60amino acid residues in length and having a bit score for the alignmentof the sequence to the regulator of chromosome condensation domain (HMM)of at least 30. Preferably, regulator of chromosome condensationdomain(s) include at least about 20 to 80 amino acids, more preferablyabout 30 to 70 amino acid residues, or about 40 to 60 amino acids andhave a bit scores for the alignment of the sequences to the regulator ofchromosome condensation domain(s) (HMM) of at least 5, 10, 20, 30 orgreater.

Regulator of chromosome condensation domains typically contain aconserved regulator of chromosome condensation signature 2 (RCC1_(—)2)pattern which participates in the catalytic mechanism. The conservedRCC1_(—)2 pattern is as follows:[LIVMFA]-[STAGC](2)-G-X(2)-H-[STAGLI]-[LIVMFA]-X-[LIVM] (SEQ ID NO:34).

In the above conserved signature sequence, and other motifs or signaturesequences described herein, the standard IUPAC one-letter code for theamino acids is used. Each element in the pattern is separated by a dash(-); square brackets ([ ]) indicate the particular residues that areaccepted at that position; x indicates that any residue is accepted atthat position; and numbers in parentheses (( )) indicate the number ofresidues represented by the accompanying amino acid.

A 50352 protein contains one, two, three, preferably four, and mostpreferably five or greater RCC1_(—)2 pattern(s) at about amino acidresidues 28 to 38, 80 to 90, 133 to 143, 186 to 196, and 241 to 251 ofSEQ ID NO:30.

In a preferred embodiment, a 50352 polypeptide or protein has one, two,three, preferably four, and most preferably five or greater “regulatorof chromosome condensation domain(s)” or one, two, three, preferablyfour and most preferably five or more region(s) which includes at leastabout 20 to 80 more preferably about 30 to 40 or 40 to 60 amino acidresidues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100%homology with a “regulator of chromosome condensation domain,” e.g., theregulator of chromosome condensation domain of human 50352 (e.g.,residues 43 to 92, 93 to 145, 146 to 198, 200 to 253, and 254 to 304 ofSEQ ID NO:30).

To identify the presence of a “regulator of chromosome condensationdomain” in a 50352 protein sequence, and make the determination that apolypeptide or protein of interest has a particular profile, the aminoacid sequence of the protein can be searched against the Pfam databaseof HMMs (e.g., the Pfam database, release 2.1) using the defaultparameters. For example, the hmmsf program, which is available as partof the HMMER package of search programs, is a family specific defaultprogram for MILPAT0063 and a score of 15 is the default threshold scorefor determining a hit. Alternatively, the threshold score fordetermining a hit can be lowered (e.g., to 8 bits). A description of thePfam database can be found in Sonhammer et al. (1997) Proteins28:405-420 and a detailed description of HMMs can be found, for example,in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al.(1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J.Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci.2:305-314, the contents of which are incorporated herein by reference. Asearch was performed against the HMM database resulting in theidentification of five “regulator of chromosome condensation domains” inthe amino acid sequence of human 50352 at about residues 43 to 92, 93 to145, 146 to 198, 200 to 253, and 254 to 304 of SEQ ID NO:30.

For further identification of domains, and make the determination that apolypeptide or protein of interest has a particular profile, the aminoacid sequence of the protein can be searched against a database ofdomains, e.g., the ProDom database (Corpet et al. (1999), Nucl. AcidsRes. 27:263-267). The ProDom protein domain database consists of anautomatic compilation of homologous domains. Current versions of ProDomare built using recursive PSI-BLAST searches (Altschul et al. (1997)Nucleic Acids Res. 25:3389-3402; Gouzy et al. (1999) Computers andChemistry 23:333-340) of the SWISS-PROT 38 and TREMBL protein databases.The database automatically generates a consensus sequence for eachdomain. A BLAST search was performed against the HMM database resultingin the identification of a “conjugation ubiquitin cyclin KIAA0032binding CG9153 cyclin E” domain in the amino acid sequence of human50352 at about residues 374 to 645 of SEQ ID NO:30, and two “ligaseubiquitin conjugation ubiquitin-protein 6.3.2” domains in the amino acidsequence of human 50352 at about residues 758 to 811 and 836 to 1013 ofSEQ ID NO:30.

A 50352 family member can include at least one homologous to the E6-APcarboxyl terminus domain and at least one, two, three, preferably four,and most preferably five or greater regulator of chromosome condensationdomain(s). Furthermore, a 50352 family member can include at least one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, and preferably fourteen protein kinase C phosphorylation sites(Prosite PS00005); at least one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty, twenty one, twenty two, twentythree, twenty four, and preferably twenty five casein kinase IIphosphorylation sites (Prosite PS00006); at least one, preferably twoN-glycosylation sites (Prosite PS00001); at least one, two, three, andpreferably four cAMP/cGMP protein kinase phosphorylation sites (PrositePS00004); at least one tyrosine kinase phosphorylation site (PrositePS00007); at least one amidation site (Prosite PS00009); at least one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,and preferably thirteen N-myristoylation sites (Prosite PS00008); atleast one conjugation ubiquitin cyclin KIAA0032 binding CG9153 cyclin Edomain (ProDom Accession Number PD136613); at least one ligase ubiquitinconjugation ubiquitin-protein 6.3.2 domain (ProDom Accession NumberPD255820); and at least one ligase ubiquitin conjugation 6.3.2 domain(ProDom Accession Number PD002225).

As the 50352 polypeptides of the invention can modulate 50352-mediatedactivities, they can be useful for developing novel diagnostic andtherapeutic agents for ubiquitin-protein ligase-associated or other50352-associated disorders, as described below.

Ubiquitin mediated intracellular proteolysis is essential for cellviability. Abnormalities within the ubiquitin pathway, either withubiquitin-protein ligases or with de-ubiquitinating enzymes, causeproblems with substrate recognition or supply of free ubiquitin,respectively. Such abnormalities can lead to or contribute to diseasepathogenesis, such as human neurodegenerative diseases. Layfield et al.,(2001) Neuropathol Appl Neurobiol 27(3): 171-179.

As used herein, a “ubiquitin-protein ligase-mediated activity” includesan activity which involves the addition of ubiquitin to eithersubstrates or ubiquitin moieties bound to substrates. This activityinvolves both the recognition of substrate specificity as well as thecreation of an isopeptide bond between glycine residues of ubiquitin andan alpha amino group of a lysine residue of the substrate or of apreviously conjugated ubiquitin. Therefore, these enzymes areresponsible for recognizing proteins which need to undergo intracellularproteolysis as well as for the attachment of ubiquitin molecules to suchproteins deemed for degradation.

As used herein, a “50352 activity”, “biological activity of 50352” or“functional activity of 50352”, refers to an activity exerted by a 50352protein, polypeptide or nucleic acid molecule on e.g., a50352-responsive cell or on a 50352 substrate, e.g., a proteinsubstrate, as determined in vivo or in vitro. In one embodiment, a 50352activity is a direct activity, such as an association with a 50352target molecule. A “target molecule” or “binding partner” is a moleculewith which a 50352 protein binds or interacts in nature. A 50352activity can also be an indirect activity, e.g., a cellular signalingactivity mediated by interaction of the 50352 protein with a 50352receptor.

Based on the above-described sequence structures and similarities tomolecules of known function, the 50352 molecules of the presentinvention can have similar biological activities as ubiquitin-proteinligase family members. For example, the 50352 proteins of the presentinvention can have one or more of the following activities: (1) theability to modulate ubiquitination of a substrate, e.g., a proteintargeted for degradation; (2) the ability to modulate substratespecificity for ubiquitination; (3) the ability to modulate cellularproliferation and/or differentiation; (4) the ability to modulateapoptosis; (5) the ability to modulate transcription and/or cell-cycleprogression; (6) the ability to modulate signal-transduction; (7) theability to modulate antigen processing; (8) the ability to modulatecell-cell adhesion; (9) the ability to modulate receptor-mediatedendocytosis; (10) the ability to modulate organelle biogenesis anddevelopment; (11) the ability to modulate neuropathological conditions;(12) the ability to modulate oncogenesis, and (13) the ability tomodulate protein levels, e.g., cellular protein levels.

The 50352 molecules of the invention can modulate the activities ofcells in tissues where they are expressed. For example, 50352 mRNA isexpressed in human umbilical vain endothelial cells, in human normalbrain cortex, in human colon tumors and in human lung tumors.Accordingly, the 50352 molecules of the invention can act as therapeuticor diagnostic agents for cardiovascular, colon, lung and neurologicaldisorders.

Thus, the 50352 molecules can act as novel diagnostic targets andtherapeutic agents for controlling one or more ubiquitin-proteinligase-associated or other 50352-associated disorders. As used herein,“ubiquitin-protein ligase disorders” are diseases or disorders whosepathogenesis is caused by, is related to, or is associated with aberrantor deficient ubiquitin-protein ligase function or expression. The 50352molecules can be used to treat neurological disorders in part becausethe 50352 mRNA is expressed in the brain.

The 50352 molecules can also be used to treat cardiovascular disordersin part because the 50352 mRNA is expressed in human umbilical veinendothelial cells.

The 50352 molecules can also be used to treat colon disorders in partbecause the 50352 mRNA is expressed in human colon tumors.

The 50352 molecules can also be used to treat lung disorders in partbecause the 50352 mRNA is expressed in human lung tumors.

The 50352 molecules and modulators thereof can act as novel therapeuticagents for controlling one or more of cellular proliferative and/ordifferentiative disorders, hormonal disorders, immune and inflammatorydisorders, neurological disorders, blood vessel disorders, plateletdisorders, cardiovascular disorders, endothelial cell disorders, liverdisorders, viral diseases, pain or metabolic disorders.

Isolation of 50352

Total RNA was prepared from various human tissues by a single stepextraction method using RNA STAT-60 according to the manufacturer'sinstructions (TelTest, Inc). Each RNA preparation was treated with DNaseI (Ambion) at 37° C. for 1 hour. DNAse I treatment was determined to becomplete if the sample required at least 38 PCR amplification cycles toreach a threshold level of fluorescence using β-2 microglobulin as aninternal amplicon reference. The integrity of the RNA samples followingDNase I treatment was confirmed by agarose gel electrophoresis andethidium bromide staining. After phenol extraction cDNA was preparedfrom the sample using the SUPERSCRIPT™ Choice System following themanufacturer's instructions (GibcoBRL). A negative control of RNAwithout reverse transcriptase was mock reverse transcribed for each RNAsample.

Gene Expression of 50352

TaqMan analysis results indicate significant 50352 expression in humanumbilical vein endothelial cells, in normal human brain cortex, in lungtumors and in colon tumors.

Human 16658

The present invention is based, in part, on the discovery of a novelhuman kinase, referred to herein as “16658”.

The human 16658 sequence (SEQ ID NO:35), which is approximately 3633nucleotides long including untranslated regions, contains a predictedmethionine-initiated coding sequence of about 3390 nucleotides(nucleotides 23-3412 of SEQ ID NO:35; 1-3390 of SEQ ID NO:37), notincluding the terminal codon. The coding sequence encodes a 1130 aminoacid protein (SEQ ID NO:36).

This mature protein form is approximately 1130 amino acid residues inlength (from about amino acid 1 to amino acid 1130 of SEQ ID NO:36).Human 16658 contains the following regions or other structural features:a predicted kinase domain located at about amino acid residues 725-1021of SEQ ID NO:36; and predicted transmembrane domains which extend fromabout amino acids 103-119 and 642-665 of SEQ ID NO:36. A hydropathy plotof human 16658 was performed. Polypeptides of the invention includefragments which include: all or part of a hydrophobic sequence, e.g.,the sequence from about amino acid 100 to 120, from about 280 to 290,and from about 985 to 650 of SEQ ID NO:36; all or part of a hydrophilicsequence, the sequence from about amino acid 50 to 80, from about 620 to640, and from about 680 to 690 of SEQ ID NO:36; a sequence whichincludes a Cys, or a glycosylation site.

The mature human 16658 protein contains the following structuralfeatures: two predicted transmembrane domains located at about aminoacids 103-119 and 642-665 of SEQ ID NO:36. Predicted transmembranedomains extend from about amino acid 103 (cytoplasmic end) to aboutamino acid 119 (extracellular end) of SEQ ID NO:36; and from about aminoacid 642 (extracellular end) to about amino acid 665 (cytoplasmic end);one extracellular loop found at about amino acid 120-641 of SEQ IDNO:36; one N-terminal cytoplasmic domain is found at about amino acidresidues 1-102 of SEQ ID NO:36; and a C-terminal cytoplasmic domain isfound at about amino acid residues 666-1130 of SEQ ID NO:36.

The 16658 protein also includes the following domains: fourN-glycosylation sites (PS00001) located at about amino acids 437-440,491-494, 504-507, and 850-853 of SEQ ID NO:36; one cAMP- andcGMP-dependent protein kinase phosphorylation site (PS00004) located atabout amino acids 945-948 of SEQ ID NO:36; sixteen predicted proteinkinase C phosphorylation sites (PS00005) located at about amino acids40-42, 83-85, 201-203, 214-216, 293-295, 304-306, 339-341, 521-523,586-588, 621-623, 666-668, 741-743, 758-760, 794-796, 1066-1068, and1115-1117 of SEQ ID NO:36; twenty-seven predicted casein kinase IIphosphorylation sites (PS00006) located at about amino 19-22, 83-86,155-158, 201-204, 214-217, 240-243, 252-255, 322-325, 333-336, 361-364,396-399, 471-474, 532-535, 628-631, 699-702, 705-708, 937-940, 949-952,965-968, 975-978, 982-985, 1022-1025, 1036-1039, 1047-1050, 1059-1062,1082-1085, and 1091-1094 of SEQ ID NO:36; three predicted tyrosinekinase phosphorylation sites (PS00007) located at about amino acids577-584, 698-706, and 881-888 of SEQ ID NO:36; eleven predictedN-myristoylation sites (PS00008) located at about amino acids 47-52,286-291, 368-373, 383-388, 467-472, 488-493, 598-603, 851-856, 871-876,994-999, and 1070-1075 of SEQ ID NO:36; two predicted amidation site(PS00009) located at about amino acids 452-455 and 747-750 of SEQ IDNO:36; one predicted protein kinases ATP-binding region signature site(PS00107) located at about amino acids 731-739 of SEQ ID NO:36; onetyrosine protein kinase specific active-site sign (PS00109) located atabout amino acids 888-900 of SEQ ID NO:36; and one receptor tyrosinekinase class V signature 1 site (PS00790) located at about amino acids286-302 of SEQ ID NO:36.

For general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420. The ephrin receptor ligand binding domain of human16658 (amino acids 128 to 301 of SEQ ID NO:36) aligns with a consensusamino acid sequence derived from a hidden Markov model (HMM) from PFAM(SEQ ID NO:44).

The protein kinase domain of human 16658 (amino acids 725 to 1021 of SEQID NO:36) aligns with a consensus amino acid sequence derived from ahidden Markov model (HMM) from PFAM (SEQ ID NO:45).

A BLAST alignment of human 16658 with a consensus amino acid sequencederived from a ProDomain “receptor kinase tyrosine-protein ephrinprecursor transferase ATP-binding phosphorylation type-A transmembrane”(Release 2001.1) shows amino acid residues 1 to 180 of the 181 aminoacid consensus sequence (SEQ ID NO:46) aligns with the “receptor kinasetyrosine-protein ephrin precursor transferase ATP-bindingphosphorylation type-A transmembrane” domain of human 16658, amino acidresidues 128 to 301 of SEQ ID NO:36.

Another BLAST alignment of human 16658 with a consensus amino acidsequence derived from a ProDomain “receptor kinase tyrosine-proteinprecursor ephrin transferase ATP-binding phosphorylation type-Atransmembrane” (Release 2001.1) shows amino acid residues 1 to 127 ofthe 128 amino acid consensus sequence (SEQ ID NO:47) aligns with the“receptor kinase tyrosine-protein precursor ephrin transferaseATP-binding phosphorylation type-A transmembrane” domain of human 16658,amino acid residues 411 to 534 of SEQ ID NO:36.

Another BLAST alignment of human 16658 with a consensus amino acidsequence derived from a ProDomain “receptor tyrosine-protein kinasetransm precursor ephrin EHK-2 kinase-2 type-A phosphorylation” (Release2001.1) shows amino acid residues 1 to 50 of the 50 amino acid consensussequence (SEQ ID NO:48) aligns with the “receptor tyrosine-proteinkinase transm precursor ephrin EHK-2 kinase-2 type-A phosphorylation”domain of human 16658, amino acid residues 790 to 839 of SEQ ID NO:36.

A BLAST alignment of human 16658 with a consensus amino acid sequencederived from a ProDomain “receptor kinase tyrosine-protein precursorephrin ATP-binding transferase phosphorylation type-A transmembrane”(Release 2001.1) shows amino acid residues 15 to 73 of the 74 amino acidconsensus sequence (SEQ ID NO:49) aligns with the “receptor kinasetyrosine-protein precursor ephrin ATP-binding transferasephosphorylation type-A transmembrane” domain of human 16658, amino acidresidues 354 to 410 of SEQ ID NO:36.

A BLAST alignment of human 16658 with a consensus amino acid sequencederived from a ProDomain “kinase tyrosine-protein repeat janus domainphosphorylation ATP-binding SH2” (Release 2001.1) shows amino acidresidues 1 to 178 of the 179 amino acid consensus sequence (SEQ IDNO:50), aligns with the “kinase tyrosine-protein repeat janus domainphosphorylation ATP-binding SH2e” domain of human 16658, amino acidresidues 843 to 1017 of SEQ ID NO:36.

A BLAST alignment of human 16658 with a consensus amino acid sequencederived from a ProDomain “receptor kinase tyrosine-protein precursorephrin transferase ATP-binding phosphorylation transmembrane type-A”(Release 2001.1) shows amino acid residues 28 to 82 of the 83 amino acidconsensus sequence (SEQ ID NO:51) aligns with the “receptor kinasetyrosine-protein precursor ephrin transferase ATP-bindingphosphorylation transmembrane type-A” domain of human 16658, amino acidresidues 668 to 723 of SEQ ID NO:36.

Human 14223

The present invention is based, in part, on the discovery of a novelhuman kinase, referred to herein as “14223”.

The human 14223 sequence (SEQ ID NO:38), which is approximately 2466nucleotides long including untranslated regions, contains a predictedmethionine-initiated coding sequence of about 1542 nucleotides(nucleotides 437-1978 of SEQ ID NO:38; 1-1542 of SEQ ID NO:40), notincluding the terminal codon. The coding sequence encodes a 514 aminoacid protein (SEQ ID NO:39).

This mature protein form is approximately 514 amino acid residues inlength (from about amino acid 1 to amino acid 514 of SEQ ID NO:39).Human 14223 contains the following regions or other structural features:a predicted kinase domain located at about amino acid residues 116-381of SEQ ID NO:39.

A hydropathy plot of human 14223 was performed. Polypeptides of theinvention include fragments which include: all or part of a hydrophobicsequence, e.g., the sequence from about amino acid 220 to 230, fromabout 285 to 295, and from about 310 to 320 of SEQ ID NO:39; all or partof a hydrophilic sequence, e.g., the sequence from about amino acid 60to 100, from about 205 to 215, and from about 400 to 460 of SEQ IDNO:39; a sequence which includes a Cys, or a glycosylation site.

The 14223 protein also includes the following domains: fourN-glycosylation sites (PS00001) located at about amino acids 95-98,213-216, 411-414, and 438-441 of SEQ ID NO:39; three cAMP- andcGMP-dependent protein kinase phosphorylation site (PS00004) located atabout amino acids 8-11, 84-87, and 271-274 of SEQ ID NO:39; twelvepredicted protein kinase C phosphorylation sites (PS00005) located atabout amino acids 10-12, 20-22, 29-31, 70-72, 134-136, 169-171, 184-186,371-373, 388-390, 459-461, 473-475, and 510-512 of SEQ ID NO:39;eighteen predicted casein kinase II phosphorylation sites (PS00006)located at about amino 4-7, 20-23, 71-74, 80-83, 134-137, 211-214,249-252, 274-277, 296-299, 326-329, 349-352, 371-374, 407-410, 412-415,420-423, 440-443, 450-453, and 459-462 of SEQ ID NO:39; two predictedN-myristoylation sites (PS00008) located at about amino acids 49-54 and383-388 of SEQ ID NO:39; one predicted protein kinases ATP-bindingregion signature site (PS00107) located at about amino acids 122-130 ofSEQ ID NO:39; one serine/threonine protein kinases active-site sign(PS00108) located at about amino acids 234-246 of SEQ ID NO:39.

For general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420.

The protein kinase domain of human 14223 (amino acids 116 to 381 of SEQID NO:39) aligns with a consensus amino acid sequence derived from ahidden Markov model (HMM) from PFAM (SEQ ID NO:52).

A BLAST alignment of human 14223 with a consensus amino acid sequencederived from a ProDomain “kinase serine/threonine-protein transferasereceptor ATP-binding 2.7.1.-tyrosine-protein phosphorylation precursor”(Release 2001.1) shows that amino acid residues 254 to 301, 136 to 235,51 to 184, 370 to 409, 6 to 85, and 344 to 370 of the 424 amino acidconsensus sequence (SEQ ID NOs:53-58) align with the “kinaseserine/threonine-protein transferase receptor ATP-binding2.7.1.-tyrosine-protein phosphorylation precursor” domains of human14223, found from amino acid residues 285 to 329, 199 to 286, 124 to245, 293 to 323, 116 to 194, and 351 to 377 of SEQ ID NO:39.

A BLAST alignment of human 14223 with a consensus amino acid sequencederived from a ProDomain “serine/threonine similar kinase kinasesserine/threonine-protein” (Release 2001.1) shows that amino acidresidues 353 to 514 and 90 to 230 of the 717 amino acid consensussequence (SEQ ID NOs:59-60) align with the “serine/threonine similarkinase kinases serine/threonine-protein” domains of human 14223, foundfrom amino acid residues 308 to 463 and 122 to 249 of SEQ ID NO:39.

Human 16002

The present invention is based, in part, on the discovery of a novelhuman kinase, referred to herein as “16002”.

The human 16002 sequence (SEQ ID NO:41), which is approximately 2711nucleotides long including untranslated regions, contains a predictedmethionine-initiated coding sequence of about 1683 nucleotides(nucleotides 198-1880 of SEQ ID NO:41; 1-1683 of SEQ ID NO:43), notincluding the terminal codon. The coding sequence encodes a 561 aminoacid protein (SEQ ID NO:42).

This mature protein form is approximately 561 amino acid residues inlength (from about amino acid 1 to amino acid 561 of SEQ ID NO:42).Human 16002 contains the following regions or other structural features:a predicted kinase domain located at about amino acid residues 128-409of SEQ ID NO:42; and a predicted transmembrane domain which extends fromabout amino acid residue 336-354 of SEQ ID NO:42.

The 16002 protein also includes the following domains: oneN-glycosylation site (PS00001) located at about amino acids 147-150 ofSEQ ID NO:42; three cAMP- and cGMP-dependent protein kinasephosphorylation site (PS00004) located at about amino acids 71-74,105-108, and 455-458 of SEQ ID NO:42; six predicted protein kinase Cphosphorylation sites (PS00005) located at about amino acids 58-60,69-71, 100-102, 160-162, 330-332, and 437-439 of SEQ ID NO:42; eightpredicted casein kinase II phosphorylation sites (PS00006) located atabout amino 26-29, 74-77, 82-85, 117-120, 419-422, 425-428, 430-433, and557-560 of SEQ ID NO:42; four predicted N-myristoylation sites (PS00008)located at about amino acids 178-183, 326-331, 515-520, and 525-530 ofSEQ ID NO:42; one predicted ATP/GTP-binding site motif A (P-loop)(PS00017) located at about amino acids 485-492; one predicted proteinkinases ATP-binding region signature site (PS00107) located at aboutamino acids 134-142 of SEQ ID NO:42; and one serine/threonine proteinkinases active-site sign (PS00108) located at about amino acids 271-283of SEQ ID NO:42.

For general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420

Polypeptides of the invention include fragments which include: all orpart of a hydrophobic sequence, e.g., the sequence from about amino acid255 to 265, from about 330 to 350, and from about 530 to 550 of SEQ IDNO:42; all or part of a hydrophilic sequence, e.g., the sequence fromabout amino acid 30 to 50, from about 170 to 185, and from about 455 to475 of SEQ ID NO:42; a sequence which includes a Cys, or a glycosylationsite.

The protein kinase domain of human 16002 (amino acids 128 to 409 of SEQID NO:42) aligns with a consensus amino acid sequence derived from ahidden Markov model (HMM) from PFAM (SEQ ID NO:61).

A BLAST alignment of human 16002 with a consensus amino acid sequencederived from a ProDomain “kinase Ca2/calmodulin-dependent phosphorylaseserine threonine hydroxyalkyl-protein B calcium/calmodulin alphaglycogen” (Release 2001.1) shows amino acid residues 1 to 80 of the 80amino acid consensus sequence (SEQ ID NO:62) aligns with the “kinaseCa2/calmodulin-dependent phosphorylase serine threoninehydroxyalkyl-protein B calcium/calmodulin alpha glycogen” domain ofhuman 16002, amino acid residues 1 to 80 of SEQ ID NO:42.

A BLAST alignment of human 16002 with a consensus amino acid sequencederived from a ProDomain “kinase Ca2/calmodulin-dependent beta alphasynthase phosphorylase serine threonine calcium/calmodulin” (Release2001.1) shows amino acid residues 1 to 61 of the 61 amino acid consensussequence (SEQ ID NO:63) aligns with the “kinase Ca2/calmodulin-dependentbeta alpha synthase phosphorylase serine threonine calcium/calmodulin”domain of human 16002, amino acid residues 403 to 463 of SEQ ID NO:42.

A BLAST alignment of human 16002 with a consensus amino acid sequencederived from a ProDomain “kinase calcium/calmodulin alpha-dependent”(Release 2001.1). shows amino acid residues 1 to 47 of the 47 amino acidconsensus sequence (SEQ ID NO:64), aligns with the “kinasecalcium/calmodulin alpha-dependent” domain of human 16002, amino acidresidues 81 to 127 of SEQ ID NO:42.

A BLAST alignment of human 16002 with a consensus amino acid sequencederived from a ProDomain “kinase Ca2/calmodulin-dependent phosphorylaseserinethreonine hydroxyalkyl-protein B calcium/calmodulin alphaglycogen” (Release 2001.1) shows amino acid residues 1 to 36 of the 42amino acid consensus sequence (SEQ ID NO:65) aligns with the “kinaseCa2/calmodulin-dependent phosphorylase serine threoninehydroxyalkyl-protein B calcium/calmodulin alpha glycogen” domain ofhuman 16002, amino acid residues 464 to 499 of SEQ ID NO:42.

The 16658, 14223, and 16002 proteins contain a significant number ofstructural characteristics in common with members of the kinase family.The term “family” when referring to the protein and nucleic acidmolecules of the invention means two or more proteins or nucleic acidmolecules having a common structural domain or motif and havingsufficient amino acid or nucleotide sequence homology as defined herein.Such family members can be naturally or non-naturally occurring and canbe from either the same or different species. For example, a family cancontain a first protein of human origin as well as other distinctproteins of human origin, or alternatively, can contain homologues ofnon-human origin, e.g., rat or mouse proteins. Members of a family canalso have common functional characteristics.

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “16658, 14223, and 16002” nucleicacid and polypeptide molecules, which play a role in or function in thetransduction of signals for cell proliferation, differentiation andapoptosis. In one embodiment, the 16658, 14223, and 16002 moleculesmodulate the activity of one or more proteins involved in cellulargrowth or differentiation, e.g., cell growth or differentiation. Inanother embodiment, the 16658, 14223, and 16002 molecules of the presentinvention are capable of modulating the phosphorylation state of 16658,14223, and 16002 molecules or one or more proteins involved in cellulargrowth or differentiation.

16022 has homology to rat calcium/calmodulin-dependent protein kinasekinase (CaMKK) alpha. As such, without being bound by theory, 16002 isexpected to be a CaMKK that mediates responses in different pain statesof BDNF, the growth factor of the neurotrophin family that isupregulated in nociceptive neurons after axotomy and CCI and releasedinto the dorsal horn of the spinal cord. CaMKK alpha phosphorylates CaMKI and IV that regulate transcription. Itself is negatively regulated byPKA. In the brain, CaMKK alpha blocks apoptosis-induced by increase ofintracellular Ca++ levels after NMDA receptor stimulation. NMDAreceptors are very important players in the modulation of pain in thespinal cord. In addition, CaMKIV, a substrate for CaMKK alpha, isphosphorylated after BDNF exposure.

As used herein, the term “protein kinase” includes a protein orpolypeptide which is capable of modulating its own phosphorylation stateor the phosphorylation state of another protein or polypeptide. Proteinkinases can have a specificity for (i.e., a specificity tophosphorylate) serine/threonine residues, tyrosine residues, or bothserine/threonine and tyrosine residues, e.g., the dual specificitykinases. As referred to herein, protein kinases preferably include acatalytic domain of about 200-400 amino acid residues in length,preferably about 250-300 amino acid residues in length, or morepreferably about 265-296 amino acid residues in length, which includespreferably 5-20, more preferably 5-15, or preferably 11 highly conservedmotifs or subdomains separated by sequences of amino acids with reducedor minimal conservation. Specificity of a protein kinase forphosphorylation of either tyrosine or serine/threonine can be predictedby the sequence of two of the subdomains (VIb and VIII) in whichdifferent residues are conserved in each class (as described in, forexample, Hanks et al. (1988) Science 241:42-52) the contents of whichare incorporated herein by reference). These subdomains are alsodescribed in further detail herein.

Protein kinases play a role in signaling pathways associated withcellular growth. For example, protein kinases are involved in theregulation of signal transmission from cellular receptors, e.g.,growth-factor receptors; entry of cells into mitosis; and the regulationof cytoskeleton function, e.g., actin bundling. Thus, the 16658, 14223,and 16002 molecules of the present invention may be involved in: 1) theregulation of transmission of signals from cellular receptors, e.g.,growth factor receptors; 2) the modulation of the entry of cells intomitosis; 3) the modulation of cellular differentiation; 4) themodulation of cell death; and 5) the regulation of cytoskeletonfunction.

Inhibition or over stimulation of the activity of protein kinasesinvolved in signaling pathways associated with cellular growth can leadto perturbed cellular growth, which can in turn lead to cellular growthrelated disorders. As used herein, a “cellular growth related disorder”includes a disorder, disease, or condition characterized by aderegulation, e.g., an upregulation or a downregulation, of cellulargrowth. Cellular growth deregulation may be due to a deregulation ofcellular proliferation, cell cycle progression, cellular differentiationand/or cellular hypertrophy.

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as 16658, 14223, and 16002 proteinand nucleic acid molecules, which comprise a family of molecules havingcertain conserved structural and functional features.

One embodiment of the invention features 16658, 14223, and 16002 nucleicacid molecules, preferably human 16658, 14223, and 16002 molecules,e.g., 16658, 14223, and 16002. The 16658, 14223, and 16002 nucleic acidand protein molecules of the invention are described in further detailin the following subsections.

A 16658, 14223, and 16002 polypeptide can include a “kinase domain” orregions homologous with a “kinase domain”.

As used herein, the term “kinase domain” includes an amino acid sequenceof about 100-400 amino acid residues in length and having a bit scorefor the alignment of the sequence to the kinase domain (HMM) of at least8. Preferably, a kinase domain includes at least about 100-350 aminoacids, more preferably about 250-300 amino acid residues, or about265-396 amino acids and has a bit score for the alignment of thesequence to the kinase domain (HMM) of at least 16 or greater. Thekinase domain (amino acids 725-1021, 116-381, and 128-409 of SEQ IDNO:36, SEQ ID NO:39, and SEQ ID NO:42, respectively) of human 16658,14223, and 16002 were aligned with a consensus amino acid sequencederived from a hidden Markov model.

In a preferred embodiment 16658, 14223, and 16002 polypeptides orproteins have a “kinase domain” or a region which includes at leastabout 200-350 more preferably about 250-300 or 265-396 amino acidresidues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%homology with an “kinase domain,” e.g., the kinase domain of human16658, 14223, and 16002 (e.g., amino acid residues 725-1021, 116-381,and 128-409 of SEQ ID NO:36, SEQ ID NO:39, and SEQ ID NO:42,respectively).

To identify the presence of a “kinase domain” in a 16658, 14223 or 16002protein sequence, and make the determination that a polypeptide orprotein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against a database of HMMs (e.g., the Pfamdatabase, release 2.1) using the default parameters. For example, thehmmsf program, which is available as part of the HMMER package of searchprograms, is a family specific default program for MILPAT0063 and ascore of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al., (1997) Proteins 28(3):405-420 and a detaileddescription of HMMs can be found, for example, in Gribskov et al.,(1990) Meth. Enzymol. 183:146-159; Gribskov et al., (1987) Proc. Natl.Acad. Sci. USA 84:4355-4358; Krogh et al., (1994) J. Mol. Biol.235:1501-1531; and Stultz et al., (1993) Protein Sci. 2:305-314, thecontents of which are incorporated herein by reference. A search wasperformed against the HMM database resulting in the identification of a“kinase domain” in the amino acid sequence of human 16658 at aboutresidues 725 to 1021 of SEQ ID NO:36; of human 14223 at about residues116 to 381 of SEQ ID NO:39; or of human 16002 at about residues 128 to409 of SEQ ID NO:42.

For further identification of domains in a 16658, 14223, and 16002protein sequence, and make the determination that a polypeptide orprotein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against a database of domains, e.g., theProDom database (Corpet et al. (1999), Nucl. Acids Res. 27:263-267). TheProDom protein domain database consists of an automatic compilation ofhomologous domains. Current versions of ProDom are built using recursivePSI-BLAST searches (Altschul S F et al. (1997) Nucleic Acids Res.25:3389-3402; Gouzy et al. (1999) 23:333-340) of the SWISS-PROT 38 andTREMBL protein databases. The database automatically generates aconsensus sequence for each domain. A BLAST search was performed againstthe HMM database resulting in the identification of a “kinase” domain(s)in the amino acid sequence of human 16658 at about residues 128 to 301,411 to 534, 790 to 839, 354 to 410, 843 to 1017, 668 to 723 of SEQ IDNO:36 having 71%, 58%, 98%, 54%, 28% and 60% identity over thoseresidues respectively; of human 14223 at about residues 285 to 329, 199to 286, 124 to 245, 293 to 323, 116 to 194, and 351 to 377 (six localalignments) of SEQ ID NO:39 having 52%, 29%, 21%, 35%, 31% and 25%identity over those residues respectively; 308 to 463 and 122 to 249(two local alignments) of SEQ ID NO:39 having 25% identity over thoseresidues; and of human 16002 at about residues 1 to 80, 403 to 463, 81to 127, or 464 to 499 of SEQ ID NO:42 having 83%, 78%, 78%, and 94%,identity over those residues respectively.

The 16658, 14223, and 16002 proteins include an ATP-binding regionsignature. Preferably, the ATP-binding region signature includes thefollowing amino acid consensus sequence having Prosite signature asPS00107, or sequences homologous thereto:[LIV]-G-{P}-G-{P}-[FYWMGSTNH]-[SGA]-{PW}-[LIVCAT]-{PD}-x-[GSTACLIVMFY]-x(5,18)-[LIVMFYWCSTAR]-[AIVP]-[LIVMFAGCKR]-K[K binds ATP] (SEQ ID NO:66). In the above conserved motif, and othermotifs described herein, the standard IUPAC one-letter code for theamino acids is used. Each element in the pattern is separated by a dash(-); square brackets ([ ]) indicate the particular residues that areaccepted at that position; x indicates that any residue is accepted atthat position; and numbers in parentheses (( )) indicate the number ofresidues represented by the accompanying amino acid. The ATP-bindingregion of 16658 is found in the C-terminal cytoplasmic domain. TheATP-binding region of 16002 is found in the N-terminal cytoplasmicdomain

A 16658 polypeptide can also include a “tyrosine protein kinase specificactive-site signature”. Preferably, the tyrosine protein kinase specificactive-site signature includes the following amino acid consensussequence having Prosite signature as PS00109, or sequences homologousthereto: [LIVMFYC]-x-[HY]-x-D-[LIVMFY]-[RSTAC]-x(2)-N-[LIVMFYC](3) [D isan active site residue] (SEQ ID NO:67). The tyrosine protein kinasespecific active-site signature for 16658 is found in the C-terminalcytoplasmic domain. A 14223 or 16002 polypeptide can also include a“serine/threonine protein kinases active-site signature”. Preferably,the serine/threonine protein kinases active-site signature includes thefollowing amino acid consensus sequence having Prosite signature asPS00109, or sequences homologous thereto:[LIVMFYC]-x-[HY]-x-D-[LIVMFY]-K-x(2)-N-[LIVMFYCT](3) [D is an activesite residue] (SEQ ID NO:68). The serine/threonine protein kinasesactive-site signature for 14223 is found in a hydrophilic region of thepolypeptide. The serine/threonine protein kinases active-site signaturefor the 16002 polypeptide is found in N-terminal cytoplasmic domain.

A 16658 polypeptide can also include a “receptor tyrosine kinase class Vsignature 1”. Preferably, the receptor tyrosine kinase class V signature1 includes the following amino acid consensus sequence having Prositesignature as PS00790, or sequences homologous thereto:F-x-[DN]-x-[GAW]-[GA]-C-[LIVM]-[SA]-[LIVM](2)-[SA]-[LV]-[KRHQ]-[LIVA]-x(3)-[KR]-C-[PSAW] (SEQ ID NO:69). A 16658polypeptide can also include a “receptor tyrosine kinase class Vsignature 2”. Preferably, the receptor tyrosine kinase class V signature2 includes the following amino acid consensus sequence having Prositesignature as PS00791, or sequences homologous thereto:C-x(2)-[DE]-G-[DEQ]-W-x(2,3)-[PAQ]-[LIVMT]-[GT]-x-C-x-C-x(2)-G-[HFY]-[EQ](SEQ ID NO:70). The receptor tyrosine kinase class V signatures 1 and 2for 16658 are found in the extracellular loop.

A 16658 polypeptide can also include a “EGF-like domain signature 2”.Preferably, the EGF-like domain signature 2 includes the following aminoacid consensus sequence having Prosite signature as PS01186, orsequences homologous thereto: C-x-C-x(2)-[GP]-[FYW]-x(4,8)-C (SEQ IDNO:71). The EGF-like domain signature 2 for 16658 is found in theextracellular loop.

A 16002 polypeptide can also include an “ATP/GTP-binding site motif A(P-loop)”. Preferably, the ATP/GTP-binding site motif A (P-loop)includes the following amino acid consensus sequence having Prositesignature as PS00017, or sequences homologous thereto:[AG]-x(4)-G-K-[ST] (SEQ ID NO:72). The ATP/GTP-binding site motif A(P-loop) for 16002 is found in the extracellular loop.

In one embodiment, a 16658 protein includes at least one and preferablytwo transmembrane domains and a 16002 protein includes at least onetransmembrane domain. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 10 to 40 amino acid residues inlength and spans the plasma membrane. Transmembrane domains are rich inhydrophobic residues, e.g., at least 50%, 60%, 70%, 80%, 90%, 95% ormore of the amino acids of a transmembrane domain are hydrophobic, e.g.,leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domainstypically have alpha-helical structures and are described in, forexample, Zagotta, W. N. et al., (1996) Annual Rev. Neurosci. 19:235-263,the contents of which are incorporated herein by reference.

In a preferred embodiment, a 16658 protein includes at least one andpreferably two transmembrane domains and a 16002 protein includes atleast one transmembrane domain or a region which includes at least orregions which include at least about 12 to 35 more preferably about 14to 30 or 15 to 25 amino acid residues or 16, 18, 20, 22, 23, 24, 25, or30 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%,or 100% homology with a “transmembrane domain,” e.g., at least onetransmembrane domain of human 16658 or 16002 (e.g., amino acid residues103-119 and 642-665 of SEQ ID NO:36, or amino acid residues 336-354 ofSEQ ID NO:42). The transmembrane domain of human 16658 and 16002 can bevisualized in a hydropathy plot as regions of about 15 to 25 amino acidswhere the hydropathy trace is mostly above the horizontal line.

To identify the presence of a “transmembrane” domain in a 16658 or 16002protein sequence, and make the determination that a polypeptide orprotein of interest has a particular profile, the amino acid sequence ofthe protein can be analyzed by a transmembrane prediction method thatpredicts the secondary structure and topology of integral membraneproteins based on the recognition of topological models (MEMSAT, Joneset al., (1994) Biochemistry 33:3038-3049).

A mature 16658 protein includes at least one, two, preferably three“non-transmembrane regions” and a mature 16002 protein includes at leastone, and preferably two “non-transmembrane regions.” As used herein, theterm “non-transmembrane region” includes an amino acid sequence notidentified as a transmembrane domain. The non-transmembrane regions in16658 or 16002 are located at about amino acids residues 1-102, 120-641,and 666-1130 of SEQ ID NO:36 or 1-335 and 356-561 of SEQ ID NO:42.

The non-transmembrane regions of 16658 include at least one preferablytwo cytoplasmic regions, and non-transmembrane regions of 16002 includeat least one cytoplasmic region. When located at the N-terminus, thecytoplasmic region is referred to herein as the “N-terminal cytoplasmicdomain.” As used herein, an “N-terminal cytoplasmic domain” includes anamino acid sequence having about 1-400, preferably about 30-75, morepreferably about 50-350, or even more preferably about 102-335 aminoacid residues in length and is located inside of a cell or within thecytoplasm of a cell. The C-terminal amino acid residue of an “N-terminalcytoplasmic domain” is adjacent to an N-terminal amino acid residue of atransmembrane domain in a 16658 or 16002 protein. For example, anN-terminal non-transmembrane domain is located at about amino acidresidues 1-102 of SEQ ID NO:36.

In a preferred embodiment, a polypeptide or protein has an N-terminalcytoplasmic domain or a region which includes at least about 5,preferably about 1 to 400, and more preferably about 1 to 350 amino acidresidues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100%homology with an “N-terminal cytoplasmic domain,” e.g., the N-terminalcytoplasmic domain of human 16658 (e.g., residues 1 to 102 of SEQ IDNO:36).

In another embodiment, a cytoplasmic region of a 16658 protein caninclude the C-terminus and can be a “C-terminal cytoplasmic domain,”also referred to herein as a “C-terminal cytoplasmic tail.” As usedherein, a “C-terminal cytoplasmic domain” includes an amino acidsequence having a length of at least about 10, preferably about 1-500,preferably about 100-490, preferably about 150-480, more preferablyabout 200-464 amino acid residues and is located inside of a cell orwithin the cytoplasm of a cell. The N-terminal amino acid residue of a“C-terminal cytoplasmic domain” is adjacent to a C-terminal amino acidresidue of a transmembrane domain in a 16658 protein. For example, aC-terminal cytoplasmic domain is located at about amino acid residues666-1130 of SEQ ID NO:36.

In a preferred embodiment, a 16658 polypeptide or protein has aC-terminal cytoplasmic domain or a region which includes at least about5, preferably about 10 to 200, and more preferably about 150 to 200amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or100% homology with a C-terminal cytoplasmic domain,” e.g., theC-terminal cytoplasmic domain of human 16658 (e.g., residues 666-1130 ofSEQ ID NO:36).

In another embodiment, a 16658 protein includes at least onenon-cytoplasmic loop. As used herein, a “non-cytoplasmic loop” includesan amino acid sequence located outside of a cell or within anintracellular organelle. Non-cytoplasmic loops include extracellulardomains (i.e., outside of the cell) and intracellular domains (i.e.,within the cell). When referring to membrane-bound proteins found inintracellular organelles (e.g., mitochondria, endoplasmic reticulum,peroxisomes microsomes, vesicles, endosomes, and lysosomes),non-cytoplasmic loops include those domains of the protein that residein the lumen of the organelle or the matrix or the intermembrane space.For example, a “non-cytoplasmic loop” can be found at about amino acidresidues 120-641 of SEQ ID NO:36.

In a preferred embodiment, a 16658 polypeptide or protein has at leastone non-cytoplasmic loop or a region which includes at least about 4,preferably about 5 to 600, more preferably about 6 to 550 amino acidresidues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100%homology with a “non-cytoplasmic loop,” e.g., at least onenon-cytoplasmic loop of human 16658 (e.g., residues 120-641 of SEQ IDNO:36).

As the 16658, 14223, and 16002 polypeptides of the invention maymodulate 16658-, 14223-, and 16002-mediated activities, they may beuseful for developing novel diagnostic and therapeutic agents for16658-, 14223-, and 16002-mediated or related disorders. Accordingly,16658, 14223, and 16002 proteins may mediate various disorders,including cellular proliferative and/or differentiative disorders, braindisorders, pain or metabolic disorders.

The 16658, 14223, and 16002 nucleic acid and protein of the inventioncan be used to treat and/or diagnose a variety of proliferativedisorders, e.g., such disorders include hematopoietic neoplasticdisorders.

Gene Expression of 16658 and 16002

TaqMan real-time quantitative RT-PCR is used to detect the presence ofRNA transcript corresponding to human 16658 or 16002 relative to a notemplate control in a panel of human tissues or cells.

It was found that the highest expression of 16658 orthologs areexpressed in normal ovary cell lines in an oncology phase II plate asshown in the following Table 12.

TABLE 12 16658 Expression in Oncology Phase II plate Mean Tissue Type16658 β 2 Mean ∂∂ Ct Expression PIT 400 Breast N 40 19.47 20.53 0 PIT372 Breast N 40 20.18 19.82 0 CHT 558 Breast N 40 19.05 20.95 0 CLN 168Breast T: IDC 40 19.61 20.39 0 MDA 304 Breast T: MD-IDC 39.36 18.5120.85 0 NDR 57 Breast T: IDC-PD 40 19.03 20.97 0 NDR 132 Breast T:IDC/ILC 40 20.78 19.22 0 CHT 562 Breast T: IDC 39.31 18.8 20.5 0 NDR 12Breast T 36 21.84 14.16 0 PIT 208 Ovary N 27.6 18.53 9.07 1.86 CHT 620Ovary N 32.58 19.47 13.11 0.11 CLN 03 Ovary T 35.49 19.43 16.07 0 CLN 17Ovary T 39.73 19.95 19.78 0 MDA 25 Ovary T 40 21.82 18.18 0 MDA 216Ovary T 37.59 20.09 17.5 0 CLN 012 Ovary T 38.48 21.16 17.32 0 MDA 185Lung N 40 19.57 20.43 0 CLN 930 Lung N 40 18.89 21.11 0 MDA 183 Lung N40 18.06 21.95 0 MPI 215 Lung T—SmC 39.92 19.05 20.88 0 MDA 259 LungT-PDNSCCL 40 19.95 20.05 0 CHT 832 Lung T-PDNSCCL 40 18.98 21.02 0 CHT911 Lung T-SCC 37.59 19.32 18.27 0 MDA 262 Lung T-SCC 37.15 22.34 14.810 CHT 211 Lung T-AC 40 19.17 20.83 0 MDA 253 Lung T-PDNSCCL 40 18.3621.64 0 NHBE 39.42 21.09 18.34 0 CHT 396 Colon N 40 23.82 16.18 0 CHT523 Colon N 36.09 18.77 17.32 0 CHT 452 Colon N 40 17.4 22.6 0 CHT 382Colon T: MD 40 18.19 21.81 0 CHT 528 Colon T: MD 40 17.99 22.01 0 CLN609 Colon T 39.51 18.77 20.73 0 CHT 372 Colon T: MD-PD 40 19.41 20.59 0NDR 217 Colon-Liver Met 39.8 18.61 21.19 0 NDR 100 Colon-Liver Met 4018.06 21.94 0 PIT 260 Liver N (female) 40 17.02 22.98 0 ONC 102Hemangioma 40 19.22 20.78 0 A24 HMVEC-Arr 37.68 18.96 18.72 0 C48HMVEC-Prol 38.31 20.68 17.62 0

As seen by these results, 16658 molecules have been found to beunderexpressed in some tumor cells, where the molecules may beinappropriately propagating either cell proliferation or cell survivalsignals. As such, activators of the 16658 molecules are useful for thetreatment of cancer, preferably ovarian cancer, and useful as adiagnostic.

It was found that the highest expression of 16658 orthologs in phase1.5.1 expression of 16658 w/β2 is found in normal brain cortex and alsoin brain hypothalamus as shown in the following Table 13.

TABLE 13 Phase 1.5.1 Expression of 16658 w/β2 Tissue Type Mean β 2 Mean∂∂ Ct Expression Artery normal 37.62 21.77 15.85 0 Aorta diseased 39.0121.77 17.23 0 Vein normal 40 19.7 20.31 0 Coronary SMC 38.19 22.4 15.790 HUVEC 36.99 20.75 16.25 0 Hemangioma 37.32 19.04 18.29 0 Heart normal38.34 29.85 8.49 0 Heart CHF 38.87 19.26 19.61 0 Kidney 34.42 20.2 14.220.0524 Skeletal Muscle 40 22 18 0 Adipose normal 39.63 20.18 19.45 0Pancreas 39.73 21.28 18.45 0 primary osteoblasts 40 20.22 19.79 0Osteoclasts (diff) 40 17.22 22.78 0 Skin normal 40 21.95 18.05 0 Spinalcord normal 35.52 20.14 15.39 0 Brain Hypothalamus normal 30.54 21.78.83 2.1974 Nerve 39.85 21.81 18.05 0 Breast normal 40 20.03 19.97 0Breast tumor 40 20.3 19.7 0 Ovary Tumor 34.32 19.86 14.46 0.0444Prostate Normal 37.01 19.34 17.66 0 Prostate Tumor 33.24 20.34 12.90.1313 Salivary glands 38.98 19.57 19.41 0 Colon normal 34.03 17.9116.13 0.0139 Colon Tumor 39.34 18.36 20.98 0 Lung normal 38.15 17.5820.57 0 Lung tumor 39.68 19.72 19.97 0 Lung COPD 39.65 28.95 10.69 0Colon IBD 40 18.16 21.84 0 Liver normal 40 19.55 20.45 0 Liver fibrosis38.11 21.5 16.61 0 Spleen normal 39.62 18.73 20.88 0 Tonsil normal 39.6316.91 22.72 0 Lymph node normal 40 18.34 21.66 0 Small intestine normal38.42 30.05 8.37 0 Skin-Decubitus 40 20.39 19.61 0 Synovium 37.49 18.9118.57 0 BM-MNC 40 18.04 21.96 0 Activated PBMC 40 17.5 22.5 0Neutrophils 40 18.4 21.61 0 Megakaryocytes 40 18.09 21.91 0 Erythroid 4021 19 0 Brain Cortex normal 28.31 23.11 5.19 27.3939 DRG (Dorsal RootGanglion) 33.59 21.65 11.94 0.2554 Ovary normal 31.93 19.76 12.17 0.217

The highest levels of expression of 16658 orthologs in expression of16658 w/β2 (Table 14) and in pain human panel phase I (Table 15) wasfound in brain. 16002 was also expressed at much lower levels in spinalcord, DRG, thymus, salivary gland and trachea.

TABLE 14 Expression of 16002 w/β2 Tissue Type Mean β 2 Mean ∂∂ CtExpression Artery normal 38.79 21.54 7.25 0 Vein normal 32.73 19.9712.76 0.1442 Aortic SMC EARLY 28.18 20.94 7.24 6.6152 Coronary SMC 28.4521.41 7.04 7.5726 Static HUVEC 28.9 20.44 8.46 2.8398 Shear HUVEC 27.9220.37 7.55 5.3361 Heart normal 28.25 18.66 9.59 1.2975 Heart CHF 29.318.34 10.96 0.502 Kidney 27.47 19.12 8.35 3.0754 Skeletal Muscle 31.721.29 10.41 0.7324 Adipose normal 32.27 19.48 12.79 0.1417 Pancreas30.33 20.44 9.89 1.0539 primary osteoblasts 30.16 19.1 11.06 0.47Osteoclasts (diff) 29.45 16.77 12.68 0.1529 Skin normal 30.22 20.68 9.541.3433 Spinal cord normal 29.13 19.47 9.66 1.2361 Brain Cortex normal23.04 20.75 2.28 205.8978 Brain Hypothalamus normal 27.03 21.25 5.7818.2621 Nerve 34.86 24.16 10.7 0.6011 DRG (Dorsal Root Ganglion) 27.0721.12 5.95 16.176 Glial Cells (Astrocytes) 28.18 21.9 6.29 12.8241Glioblastoma 27.87 17.25 10.62 0.6354 Breast normal 29.34 19.52 9.821.1063 Breast tumor 26.34 17.91 8.43 2.8995 Ovary normal 29.27 19.8 9.461.415 Ovary Tumor 30.86 19.22 11.64 0.3133 Prostate Normal 27.75 18.958.79 2.2513 Prostate Tumor 25.32 16.34 8.97 1.9873 Epithelial Cells(Prostate) 29.62 20.36 9.26 1.631 Colon normal 28.68 17.66 11.02 0.4816Colon Tumor 25.57 18.23 7.34 6.1936 Lung normal 30.5 17.28 13.23 0.1044Lung tumor 26.75 17.41 9.34 1.5484 Lung COPD 28.23 17.9 10.33 0.7769Colon IBD 29.56 16.81 12.75 0.1452 Liver normal 32.91 19.06 13.85 0.0677Liver fibrosis 31.59 20.67 10.91 0.5179 Dermal Cells-fibroblasts 28.2619.29 8.97 1.9942 Spleen normal 29.64 19.12 10.52 0.681 Tonsil normal27.81 16.56 11.25 0.4106 Lymph node 29.51 18.22 11.3 0.398 Resting PBMC30.27 19.47 10.8 0.5628 Skin-Decubitus 28.75 20.12 8.63 2.5329 Synovium28.93 18.57 10.36 0.7635 BM-MNC (Bone marrow 26.46 16.51 9.95 1.011mononuclear cells) Activated PBMC 28.18 15.37 12.82 0.1388

TABLE 15 16002 Human Panel Phase I Tissue Type 16002 β2.803 ∂CtExpression Adrenal Gland 25.91 17.60 8.31 3.15 Brain 23.20 19.95 3.26104.75 Heart 28.94 18.08 10.86 0.54 Kidney 27.86 17.86 10.00 0.98 Liver29.25 18.08 11.17 0.44 Lung 28.16 16.28 11.88 0.27 Mammary Gland 27.3817.24 10.14 0.89 Pancreas 28.21 20.37 7.85 4.35 Placenta 33.31 18.1815.13 0.03 Prostate 30.26 16.90 13.36 0.10 Salivary Gland 26.38 18.497.89 4.22 Muscle 29.10 19.92 9.18 1.72 Sm. Intestine 27.59 18.05 9.541.35 Spleen 30.06 15.99 14.07 0.06 Stomach 28.18 17.64 10.55 0.67 Teste25.67 19.68 5.99 15.79 Thymus 26.28 17.56 8.72 2.37 Trachea 27.28 18.418.88 2.13 Uterus 27.08 18.54 8.54 2.69 Spinal Cord 28.49 18.69 9.80 1.12DRG 28.20 19.24 8.96 2.01 Skin 28.03 18.08 9.95 1.01

The following Table 16 showing the results of a TaqMan experiment withrat panel phase I, showed a similar pattern of expression as that of thehuman 16002 gene. This gene is also expressed in the SCG.

TABLE 16 12818 Rat Panel Phase I Tissue r12818 18S ∂Ct Expression Brain23.92 12.85 11.07 0.47 Spinal Cord 23.77 12.86 10.91 0.52 DRG 25.2713.69 11.59 0.33 SCG 26.10 13.58 12.52 0.17 Hairy Skin 26.98 13.91 13.070.12 Gastro Muscle 28.88 14.24 14.64 0.04 Heart 29.76 13.68 16.08 0.01Kidney 28.67 13.43 15.25 0.03 Liver 30.95 12.95 18.00 0.00 Lung 25.9112.71 13.20 0.11 Spleen 27.52 13.78 13.75 0.07 Aorta 28.78 14.20 14.580.04 Adrenal Gland 25.46 13.35 12.12 0.23 Salivary Gland 27.47 13.1514.33 0.05 Thyroid 26.47 14.37 12.10 0.23 Prostate 28.93 13.50 15.430.02 Thymus 26.64 13.56 13.08 0.12 Trachea 28.04 14.37 13.67 0.08Esophagus 27.12 14.16 12.96 0.13 Duodenum 31.70 14.48 17.22 0.01Diaphragm 29.63 13.98 15.65 0.02

The following table, Table 17, shows the results of a TaqMan experimentwith rat phase II and II panels. There is little or no regulation ofthis gene in animal models.

TABLE 17 12818 Rat Panel Phase II and III Tissue r12818 18S ∂CtExpression Naïve DRG 24.77 12.27 12.50 0.17 I DRG CCI 3 24.51 12.5211.99 0.25 I DRG CCI 7 25.22 12.03 13.20 0.11 I DRG CCI 10 25.25 12.0913.16 0.11 I DRG CCI 14 25.07 11.98 13.09 0.11 I DRG CCI 28 25.33 11.9913.34 0.10 Naïve DRG 25.17 12.34 12.83 0.14 I DRG CFA 1 24.92 12.0812.84 0.14 I DRG CFA 3 25.17 12.23 12.94 0.13 I DRG CFA 7 25.05 12.1612.89 0.13 I DRG CFA 10 24.88 12.34 12.54 0.17 I DRG CFA 14 24.57 11.8412.73 0.15 I DRG CFA 28 24.69 12.14 12.55 0.17 Naïve DRG 24.99 11.8213.18 0.11 I DRG AXT 1 24.95 12.11 12.84 0.14 I DRG AXT 3 25.24 12.3012.94 0.13 I DRG AXT 7 25.57 12.15 13.43 0.09 I DRG AXT 14 25.27 11.9913.29 0.10 Naïve SC 22.31 12.34 9.97 1.00 I SC CCI 3 22.80 12.63 10.170.87 I SC CCI 7 22.71 12.79 9.92 1.03 I SC CCI 10 22.42 13.14 9.28 1.61I SC CCI 14 22.33 12.12 10.22 0.84 I SC CCI 28 22.47 13.07 9.40 1.48Naïve SC 22.67 12.85 9.82 1.11 I SC CFA 1 22.96 12.77 10.19 0.86 I SCCFA 3 22.75 12.45 10.31 0.79 I SC CFA 7 23.30 12.37 10.93 0.51 I SC CFA10 23.09 12.86 10.23 0.83 I SC CFA 14 23.14 13.30 9.84 1.09 I SC CFA 2823.03 12.97 10.06 0.94 Naïve SC 22.75 12.71 10.04 0.95 I SC AXT 1 22.6512.81 9.85 1.09 I SC AXT 3 23.26 12.51 10.75 0.58 I SC AXT 7 23.36 12.4210.94 0.51 I SC AXT 14 22.41 13.34 9.07 1.86 mAorta 38.01 14.41 23.600.00 mL4/5 DRG 34.11 13.69 20.42 0.00 mCerv DRG 34.00 14.01 19.99 0.00mL4/5 SC 28.77 12.79 15.98 0.02 mCerv. SC 28.95 12.95 16.00 0.02mSciatic 39.37 14.58 24.80 0.00 mPancreas 40.00 28.96 11.04 0.47 SNS WT32.44 13.23 19.21 0.00 SNS WT 31.22 12.66 18.56 0.00 rDRG 26.62 13.6412.99 0.12 rSC 28.40 15.21 13.19 0.11In situ Hybridization of 16002

ISH experiment using a human probe showed that the 16002 gene isexpressed in human, monkey and rat cortex as well as in monkey and ratspinal cord and DRG. In the rat brain this gene is also expressed athigh levels in the hippocampus and at lower levels in the thalamus andin the basal ganglia. In the spinal cord, 16002 is expressed in laminaII of the dorsal horn as well as in laminae V-X. In the DRG, neuronsmainly of intermediate size are expressing this gene.

Human 50566 (G2RF)

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “Glyoxalase II Related Factor “or“G2RF” or “50566” nucleic acid and polypeptide molecules, which arenovel members of the glyoxalase system enzyme family. These novelmolecules are capable of metabolizing toxic compounds (e.g., cytotoxinor other metabolites) in a cell, e.g., a heart, placenta, lung, liver,skeletal muscle, thymus, kidney, pancreas, testis, ovary, prostate,colon, or brain cell. By doing so, these molecules help maintain aproper equilibrium of toxic compounds in a cell, thus preventing theoccurrence of cellular damage.

As used herein, a “glyoxalase II related factor” includes a protein orpolypeptide which is involved in the metabolism of cytotoxins and othermetabolites, as well as in the regulation of their cellular levels. Asused herein, the terms “cytotoxins” and “metabolites” include compoundswhich can be harmful or detrimental to a cell when present in sufficientconcentrations or quantities. Cytotoxins and metabolites include thosewhich arise from endogenous sources, e.g., the normal metabolicprocesses of the cell such as the energetic metabolic pathways.Cytotoxins and metabolites may also enter the cell from theextracellular milieu. Cytotoxins and metabolites which enter the cellinclude those which originate from outside the organism (xenobioticcompounds). Examples of cytotoxins and metabolites includeoxaloaldehydes, hydrocarboxylic acids, pharmacological compounds (e.g.,chemotherapeutic compounds and anti-cancer drugs), oxidative compounds,glutathione-conjugates, energy metabolites, methylglyoxal, and the like.

As used herein, the phrase “regulation of cellular levels” includescellular mechanisms involved in regulating and influencing the levels(e.g., intracellular and/or extracellular levels) of cytotoxins andmetabolites (e.g., oxaloaldehydes and hydrocarboxylic acids orglutathione-conjugates). Such mechanisms include the conversion ofpotentially cytotoxic compounds into non-toxic or less toxic compounds,e.g., conversion of oxaloaldehydes (such as methylglyoxal or glutathioneconjugates) into hydrocarboxylic acids (such as lactate) in response tobiological cues, such as formation of nucleotide adjunct, modificationof amino acids, and oxidative stress. The maintenance of regulation ofcytotoxin and metabolite levels is particularly important for a cell'sability to function properly. Thus, the G2RF or 50566 molecules, byparticipating in the regulation of cytotoxin and metabolite levels, mayprovide novel diagnostic targets and therapeutic agents for controllingcytotoxin- and metabolite-associated disorders (e.g.,glyoxalase-associated disorders, oxaloaldehyde- andmethylglyoxal-associated disorders).

As used herein, the terms “cytotoxin-associated disorders” and“metabolite-associated disorders” include disorders, diseases, orconditions which are characterized by aberrant, e.g., upregulated,downregulated, or misregulated, cytotoxin and/or metabolite levels(e.g., oxaloacetate, hydroxycarboxylic acid, thioester compound, orglutathione-conjugated compound levels). Examples of such disorders mayinclude cardiovascular disorders, e.g., arteriosclerosis, ischemiareperfusion injury, restenosis, arterial inflammation, vascular wallremodeling, ventricular remodeling, rapid ventricular pacing, coronarymicroembolism, tachycardia, bradycardia, pressure overload, aorticbending, coronary artery ligation, vascular heart disease, atrialfibrillation, long-QT syndrome, congestive heart failure, sinus nodedysfunction, angina, heart failure, hypertension, atrial fibrillation,atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy,myocardial infarction, coronary artery disease, coronary artery spasm,or arrhythmia.

Other examples of cytotoxin- and metabolite-associated disorders includedisorders of the central nervous system, e.g., cystic fibrosis, type 1neurofibromatosis, cognitive and neurodegenerative disorders, examplesof which include, but are not limited to, Alzheimer's disease, dementiasrelated to Alzheimer's disease (such as Pick's disease), Parkinson's andother Lewy diffuse body diseases, senile dementia, Huntington's disease,Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophiclateral sclerosis, progressive supranuclear palsy, epilepsy, andCreutzfeldt-Jakob disease; autonomic function disorders such ashypertension and sleep disorders, and neuropsychiatric disorders, suchas depression, schizophrenia, schizoaffective disorder, korsakoff'spsychosis, mania, anxiety disorders, or phobic disorders; learning ormemory disorders, e.g., amnesia or age-related memory loss, attentiondeficit disorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. Furthercytotoxin- and metabolite-associated disorders include, for example,those listed in the American Psychiatric Association's Diagnostic andStatistical manual of Mental Disorders (DSM), the most current versionof which is incorporated herein by reference in its entirety.

Still other examples of cytotoxin- and metabolite-associated disordersinclude cellular proliferation, growth, differentiation, or migrationdisorders. Cellular proliferation, growth, differentiation, or migrationdisorders include those disorders that affect cell proliferation,growth, differentiation, or migration processes. As used herein, a“cellular proliferation, growth, differentiation, or migration process”is a process by which a cell increases in number, size or content, bywhich a cell develops a specialized set of characteristics which differfrom that of other cells (e.g., spermatogenesis), or by which a cellmoves closer to or further from a particular location or stimulus. Suchdisorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumorangiogenesis and metastasis; skeletal dysplasia; hepatic disorders; andhematopoietic and/or myeloproliferative disorders.

Still other examples of cytotoxin- and metabolite-associated disordersinclude disorders of the immune system, such as the immune responseduring starvation, Wiskott-Aldrich syndrome, viral infection, autoimmunedisorders or immune deficiency disorders, e.g., congenital X-linkedinfantile hypogammaglobulinemia, transient hypogammaglobulinemia, commonvariable immunodeficiency, selective IgA deficiency, chronicmucocutaneous candidiasis, or severe combined immunodeficiency. Otherexamples of cytotoxin- and metabolite-associated disorders includecongenital malformities, including facio-genital dysplasia; and skindisorders, including microphthalmia with linear skin defects syndrome.

The term “family” when referring to the polypeptide and nucleic acidmolecules of the invention is intended to mean two or more polypeptidesor nucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. For example, the family of G2RF polypeptides comprise at leastone “transmembrane domain.” As used herein, the term “transmembranedomain” includes an amino acid sequence of about 20-45 amino acidresidues in length which spans the plasma membrane. More preferably, atransmembrane domain includes about at least 20, 25, 30, 35, 40, or 45amino acid residues and spans the plasma membrane. Transmembrane domainsare rich in hydrophobic residues, and typically have an alpha-helicalstructure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%,95% or more of the amino acids of a transmembrane domain arehydrophobic, e.g., leucines, isoleucines, alanines, valines,phenylalanines, prolines or methionines. Transmembrane domains aredescribed in, for example, Zagotta W. N. et al., (1996) Annual Rev.Neurosci. 19: 235-263, the contents of which are incorporated herein byreference. Amino acid residues 129-145 of the human G2RF polypeptide(SEQ ID NO:74) comprise a transmembrane domain. Accordingly, G2RF or50566 polypeptides having at least 50-60% homology, preferably about60-70%, more preferably about 70-80%, or about 80-90% homology with atransmembrane domain of human G2RF or 50566 are within the scope of theinvention.

To identify the presence of a transmembrane domain in a G2RF or 50566protein, and make the determination that a protein of interest has aparticular profile, the amino acid sequence of the protein may besubjected to MEMSAT analysis. A MEMSAT analysis resulted in theidentification of a transmembrane domain in the amino acid sequence ofhuman G2RF or 50566 (SEQ ID NO:74) at about residues 129-145.

In another embodiment, a G2RF or 50566 molecule of the present inventionis identified based on the presence of at least one“metallo-beta-lactamase superfamily domain”, also referred tointerchangeably herein as a “lactamase-B domain.” As used herein, theterm “metallo-beta-lactamase superfamily domain” or “lactamase-B domain”includes a protein domain having an amino acid sequence of about 80-250amino acid residues and has a bit score of at least 80 when comparedagainst a metallo-beta-lactamase superfamily domain Hidden Markov Model(HMM). Preferably, a “metallo-beta-lactamase superfamily domain” has anamino acid sequence of about 90-240, 100-220, 120-200, 140-180, or morepreferably, about 165 amino acid residues, and a bit score of at least90, 100, 110, 120, or more preferably about 133.3. In a preferredembodiment, a “metallo-beta-lactamase superfamily domain” includes adomain which has an amino acid sequence of about 80-250 amino acidresidues, and serves to catalyze the hydrolysis of a thioester (e.g. thethioester in a lactoylglutathione compound). Metallo-beta lactamasesuperfamily domains are described in, for example, Carfi et al., (1995)EMBO Journal 14:4914-4921, the contents of which are incorporated hereinby reference. To identify the presence of a metallo-beta-lactamasesuperfamily domain in a G2RF protein, and make the determination that aprotein of interest has a particular profile, the amino acid sequence ofthe protein may be searched against a database of known protein domains(e.g., the HMM database). The metallo-beta-lactamase superfamily domainhas been assigned the PFAM Accession No. PF00753 and InterPro AccessionNo. IPR001279. A search was performed against the HMM database resultingin the identification of a metallo-beta-lactamase superfamily domain inthe amino acid sequence of human G2RF or 50566 (SEQ ID NO:74) at aboutresidues 7-172 of SEQ ID NO:74.

A description of the Pfam database can be found in Sonhammer et al.(1997) Proteins 28:405-420 and a detailed description of HMMs can befound, for example, in Gribskov et al. (1990) Meth. Enzymol.183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference.

In a preferred embodiment, the G2RF or 50566 molecules of the inventioninclude at least one transmembrane domain and/or at least one ametallo-beta-lactamase superfamily domain.

Isolated G2RF or 50566 polypeptides of the present invention, have anamino acid sequence sufficiently identical to the amino acid sequence ofSEQ ID NO:74 or are encoded by a nucleotide sequence sufficientlyidentical to SEQ ID NO:73 or 75. As used herein, the term “sufficientlyidentical” refers to a first amino acid or nucleotide sequence whichcontains a sufficient or minimum number of identical or equivalent(e.g., an amino acid residue which has a similar side chain) amino acidresidues or nucleotides to a second amino acid or nucleotide sequencesuch that the first and second amino acid or nucleotide sequences sharecommon structural domains or motifs and/or a common functional activity.For example, amino acid or nucleotide sequences which share commonstructural domains having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identityacross the amino acid sequences of the domains and contain at least oneand preferably two structural domains or motifs, are defined herein assufficiently identical. Furthermore, amino acid or nucleotide sequenceswhich share at least 50%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity andshare a common functional activity are defined herein as sufficientlyidentical.

In a preferred embodiment, a G2RF or 50566 polypeptide includes at leastone or more of the following domains: a transmembrane domain and/or ametallo-beta-lactamase superfamily domain, and has an amino acidsequence at least about 50%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous or identicalto the amino acid sequence of SEQ ID NO:74. In yet another preferredembodiment, a G2RF polypeptide includes at least one or more of thefollowing domains: a transmembrane domain and/or ametallo-beta-lactamase superfamily domain, and is encoded by a nucleicacid molecule having a nucleotide sequence which hybridizes understringent hybridization conditions to a complement of a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:73 or SEQ IDNO:75. In another preferred embodiment, a G2RF polypeptide includes atleast one or more of the following domains: a transmembrane domainand/or a metallo-beta-lactamase superfamily domain, and has a G2RFactivity.

As used interchangeably herein, “G2RF or 50566 activity”, “biologicalactivity of G2RF or 50566” or “functional activity of G2RF or 50566”,includes an activity exerted by a G2RF polypeptide or nucleic acidmolecule on a G2RF responsive cell or tissue, or on a G2RF polypeptidesubstrate, as determined in vivo, or in vitro, according to standardtechniques. In one embodiment, a G2RF activity is a direct activity,such as an association with a G2RF-target molecule. As used herein, a“target molecule” or “binding partner” is a molecule with which a G2RFpolypeptide binds or interacts in nature, such that G2RF-mediatedfunction is achieved. A G2RF target molecule can be a non-G2RF molecule,for example, a non-G2RF polypeptide. In an exemplary embodiment, a G2RFtarget molecule is a G2RF ligand, e.g., a cytotoxin, a metabolite,glutathione, a gluathione-conjugated compound such aslactoylglutathione, or a thioester-containing compound. For example, aG2RF target molecule can have one or more of the following activities:(1) it may interact with cytotoxins and metabolites (e.g.,lactoylglutathione, a glutathione-conjugated metabolite, ahydroxycarboxylic acid, and the like), (2) it may catalyze themebobolism of a cytotoxin or metabolite (e.g., lactoylglutathione, aglutathione-conjugated metabolite, a hydroxycarboxylic acid, and thelike), (3) it may hydrolyze a thioester containing compound (e.g.,lactoylglutathione, and the like), (4) it may catalyze the formation ofa thioester conjugation on a substrate (e.g., lactate or ahydroxycarboxylic acid). Moreover, a G2RF activity is an indirectactivity, such as a cellular signaling activity mediated by interactionof the G2RF polypeptide with a G2RF ligand. The biological activities ofG2RF are described herein. For example, the G2RF polypeptides of thepresent invention can have one or more of the following activities: (1)modulation of signal transduction in a cell, (2) modulation of cytotoxinand/or metabolite levels (e.g., detoxification), (3) maintenance ofequilibrium of cytotoxins and/or metabolites, (4) modulation of canceror tumor progression, (5) modulation of cellular proliferation, (6)modulation of tissue development (e.g. embryogenesis), (7) modulation ofdifferentiation, (8) modulation of apoptosis, and (9) modulation ofenergy metabolism.

The human G2RF or 50566 cDNA sequence (SEQ ID NO:73), which isapproximately 1154 nucleotide residues long including un-translatedregions, contains a predicted methionine-initiated coding sequence ofabout 846 nucleotide residues, (nucleotide residues 22-867 of SEQ IDNO:73; 1-846 SEQ ID NO:75), not including the terminal codon. The codingsequence encodes a 282 amino acid protein having the amino acid sequenceSEQ ID NO:74.

Analysis of the Human G2RF or 50566 Molecules

A search using the polypeptide sequence of SEQ ID NO:74 was performedagainst the HMM database in PFAM resulting in the identification of ametallo-beta-lactamase superfamily domain in the amino acid sequence ofhuman G2RF or 50566 at about residues 7-172 of SEQ ID NO:74(score=133.3).

A search using the polypeptide sequence of SEQ ID NO:74 was alsoperformed against the Memsat database, resulting in the identificationof a potential transmembrane domain in the amino acid sequence of humanG2RF or 50566 (SEQ ID NO:74) at about residues 129-145, and theidentification of a potential signal peptide in the amino acid sequenceof human G2RF at about residues 1-54 of SEQ ID NO:74.

Further domain motifs were identified by using the amino acid sequenceof 50566 (SEQ ID NO:74) to search the ProDom database. Numerous matchesagainst protein domains described as “Hydrolase IIHydroxyacylglutathione Glyoxalase Glx Zinc Cytoplasmic Plasmid PeptideMultigene”, Hydrolase Similar Flavoprotein Rv2260 TuberculosisMycobacterium PH1213”, “Hydrolase II Hydroxyacylglutathione ZincGlyoxalase Glx Precursor Family”, “II Hydrolase Glyoxalase GlxHydroxyacylglutathione Zinc Precursor Specific MNCB-5687 Peptide” andthe like were identified.

A search was also performed against the Prosite database, which resultedin the identification of a potential “cAMP- and cGMP-dependent proteinkinase phosphorylation site” at residues 232-235 of SEQ ID NO:74(Prosite accession number PS00004), two potential “Protein kinase Cphosphorylation sites” at residues 86-88 and 235-237 of SEQ ID NO:74(Prosite accession number PS00005), multiple potential “Casein kinase IIphosphorylation sites” at residues 143-146, 155-158, 177-180 and 213-216of SEQ ID NO:74 (Prosite accession number PS00006), and multiplepotential N-myristoylation sites at residues 44-49, 140-145 and 274-279of SEQ ID NO:74 (Prosite accession number PS00008).

The amino acid sequence of human G2RF or 50566 was analyzed using theprogram PSORT to predict the localization of the proteins within thecell. This program assesses the presence of different targeting andlocalization amino acid sequences within the query sequence. The resultsof the analyses show that human G2RF may be localized to the cytoplasm,nucleus, mitochondria, or golgi.

Tissue Expression Analysis of G2RF mRNA Using Taqman Analysis

The following describes the tissue distribution of human G2RF mRNA in avariety of cells and tissues, as determined using the TaqMan™ procedure.

An array of human tissues were tested. Expression was greatest in thebrain cortex and hypothalamus, normal skin, heart with coronary heartfailure (CHF) and erythroid cells. Expression was also high in thekidney, coronary smooth muscle cells (SMC), human umbilical veinepithelial cells (HUVEC), normal spinal cord tissue, dorsal rootganglions and colon tumor.

Human 65552

The present invention is based, in part, on the discovery of a novelgene encoding an MMP (“MMP” is used interchangeably herein with “matrixmetalloprotease”, “matrix metalloprotease-ADAMTS” and “matrixmetalloproteinase”), the gene referred to herein as “65552”.

The human 65552 cDNA sequence (SEQ ID NO:76), which is approximately2853 nucleotide residues long including un-translated regions, containsa predicted methionine-initiated coding sequence of about 2850nucleotide residues, (i.e., nucleotide residues 1-2850 of SEQ ID NO:76;also shown in SEQ ID NO:78). The coding sequence encodes a 950 aminoacid protein having the amino acid sequence SEQ ID NO:77.

A hydropathy plot of human 65552 was performed. Polypeptides of theinvention include fragments which include: all or part of a hydrophobicsequence, e.g., the sequence from about amino acid 408 to 424 of SEQ IDNO:77; a sequence which includes a Cys, or a glycosylation site.

The 65552 protein contains a significant number of structuralcharacteristics in common with members of the matrix metalloproteinase(MMP) family. The 65552 protein contains a significant number ofstructural characteristics in common with members of the adamalysins(reprolysin) family. For example, the 65552 molecule may contain apeptidase M12B propeptide domain, a reprolysin domain, and athrombospondin domain.

Matrix metalloproteinase adamalysin molecules require a metal forcatalyzing the cleavage of peptides or proteins. Typically, theseproteases require zinc for the catalysis, ligands for which can behistidine residues. Many of these proteins have collagenase-likecatalytic activity. For example, collagens are extracellular matrixproteins responsible for the architecture and structural integrity ofmost tissues and stromelysin, which degrade the extracellular matrix.

Metalloproteases (also referred to herein as “metallopeptidases” or“metalloproteinases” or “MMPs”) are a group of highly diverse, widelydistributed proteolytic enzymes that depend on bound Ca²⁺ or Zn²⁺ foractivity. Certain metalloproteases can readily utilize Mn²⁺ and Mg²⁺ aswell. About 30 families of metalloproteases are recognized, about halfof which comprise enzymes containing the HEXXH motif (SEQ ID NO:84)(Rawlings et al. (1995) Meth Enzymol 248:183-228). The most thoroughlycharacterized of the metalloproteases is thermolysin, a member of the M4metalloprotease family.

Another metalloprotease family, the M12 family, contains the reprolysin(M12B) subfamily, which contains the snake venom metalloproteases andadamalysins family. The reprolysin subfamily also includes BRCA1, ahuman breast cancer-associated protein, and mammalian fertilin.

The ADAM subfamily of reprolysins comprises a broad family ofmultifunctional proteins, members of which may include, but are notlimited to, a disintegrin and/or a metalloprotease domain (Wolfsberg etal. (1995) Developmental Biol 169:378-383; Wolfsberg et al. (1995) JCell Biol 131:275-278; Hurskainen et al. (1999) J Biol Chem274:25555-25563).

The ADAMs are expressed by a wide variety of cell types, and areinvolved in functions as diverse as sperm-egg binding, myotubeformation, neurogenesis, and proteolytic processing of cell surfaceproteins. Their functions involve proteolysis on the cell surface: theformation and inactivation of regulatory peptides and growth factors, aswell as modification of cell surface proteins. Most members of thisfamily are snake venom endopeptidases, but there are also some mammalianproteins such as fertilin and TACE. (Fertilin is involved in sperm-eggbinding, and TACE is a member of the ADAM family that cleavesmembrane-bound TNF-alpha to generate soluble TNF-alpha). The activeenzymes degrade components of the extracellular matrix, playing a rolein the initial steps of tissue remodeling during morphogenesis, woundhealing, angiogenesis and tumor invasion.

The ADAMTSs (“a disintegrin and metalloprotease with thrombospondingtype I motifs”) subfamily of ADAMs are similar in domain organization(though different from other ADAM family members), having one or more ofthe following domains: a signal peptide, peptidase M12B propeptidedomain, a zinc binding region signature a domain, a cysteine-richdomain, a thrombospondin type-1 domain, and in many cases amembrane-spanning region and a cytoplasmic domain with signalingpotential. For example, ADAMTS-1 differs from other ADAM family membersdue to a lack of the cysteine rich, EGF and transmembrane domains andthe addition of thrombospondin type I motifs (Kuno et al. (1997) J BiolChem 272: 556-562).

Reprolysin (M12B) is a zinc metalloprotease family member with nodisintegrin-like domain, but with one propeptide for members of thepeptidase family M12, and one thrombospondin type I motif. Theseproteins have collagenase-like catalytic activity. For example,collagens are extracellular matrix proteins responsible for thearchitecture and structural integrity of most tissues. They aresynthesized as procollagens and go through a series ofpost-translational modifications both inside and outside the cell beforethey are fully functional (Duance and Bailey (1981) Handbook ofInflammation. Vol. 3 Tissue Regeneration and Repair, ed. Glynn, LEElsevier, Amsterdam, 51-109). Included in collagen maturation are thesteps of removing the extension peptides from the N- and C-termini. Thefailure of this process results in many connective tissue disorders, forexample, Ehlers-Danlos syndrome type VIIC, characterized by theretention of the N-terminal propeptide of collagen I (Lenaers et al.(1971) Eur J Biochem 23: 533-543) by a metalloprotease, procollagen IN-protease (Colige et al. (1999) Am. J. Hum. Genet. 65: 308-317).

The known members of the reprolysin subfamily mostly lack essentialpeptidase active sites, but can contain one of the following domains: aC-terminal disintegrin-like domain, an epidermal growth factor(EGF)-like domain, and transmembrane domains (Rawlings et al. (1995)Meth Enzymol 248:183-228). In addition, members of the reprolysin (M12B)subfamily of zinc metalloproteases contain a reprolysin propeptideregion. Many MMPs are expressed as latent pro-enzymes that are activatedby proteolytic cleavage. Cleavage of the M12B propeptide triggers aconformational change in the propeptide, thereby converting an M12Bfamily type metalloprotease to its active strate. Coordination of Zn2+in the active site of the catalytic domain of the M12B propeptide by acysteine residue in the prodomain is critical for inhibition of theprotease. (Overgaard et. al. (1999), J Biol. Chem, 7:274(19):13427-33).

The reprolysin domain is characteristic of extracellularmetalloproteinases, such as collagenase and stromelysin, which degradethe extracellular matrix. The members of this family are enzymes thatcleave peptides. These proteases require zinc for catalysis, ligands forwhich can be histidine residues. Members of this family are also knownas adamalysins. There are two subfamilies of adamalysins: the snakevenom metalloproteases (SVMPs) and the ADAMs (“a disintegrin andmetalloprotease”). At least 23 ADAMs have been identified to date.Members of the ADAMs family of proteins include, but are not limited to,MDC (ADAMI), ADAMTS-1, fertilin (ADAM2), cryitestin (ADAM3), epididymalapical protein I, meltrin, MS2, TNF-a converting enzyme, Kuzbanian andmetargidin.

The thrombospondin type I (TSP I) motifs in ADAMTS-1 enable it to bindto the extracellular matrix (Kuno and Matsushima (1998) J Biol Chem 273:13912-13917). TSP I motifs are conserved domains in thrombospondin 1 and2, multifunctional secretory glycoproteins involved in blood clotting,inhibiting angiogenesis and regulating the proliferation, adhesion andmigration of normal and tumor cells. The biological activities ofthrombospondin 1 and 2 are mediated by the binding of the TSP type Imotifs to extracellular matrix molecules, such as heparan sulfate,proteoglycans, fibronectin, laminin and collagen. Thrombospondin-1 is aplatelet-derived glycoprotein that is released from platelet alphagranules in response to thrombin stimulation. It is involved in celladhesion and modulates cell movement, cell proliferation, neuriteoutgrowth and angiogenesis. In general, the biological functions ofmetalloproteases include protein maturation, degradation of proteins,such as extracellular matrix proteins (ECM proteins), tumor growth,metastasis and angiogenesis, among others. Thus, metalloproteases arelikely to play important roles in a wide range of diseases including,but not limited to, cancer, arthritis, Alzheimer's disease, and avariety of inflammatory conditions. Other normal and pathologicalprocesses in which MMP-catalyzed changes in ECM protein structures havebeen implicated are described, for example, in Nagase et al. (1999) J.Biol. Chem. 274:21491-21494. Accordingly, metalloproteases are animportant target for drug action and development. Therefore, it isvaluable to the field of pharmaceutical development to identify andcharacterize previously unknown metalloproteases.

A human 65552 polypeptide can include a reprolysin domain.

In a preferred embodiment, the reprolysin domain is characteristic ofextracellular metalloproteinases.

As used herein, the term “reprolysin domain” refers to a protein domainhaving an amino acid sequence of about 100-300 amino acid residues inlength, and having an E value of about 2.5e-8. Preferably, thereprolysin domain has a length of about 100-300 amino acid residues inlength, preferably about 150-250 amino acid residues in length, morepreferably about 175-225 amino acid residues in length, and even morepreferably about 205-220 amino acids residues in length, and has anE-value of about 2.5 e-9 or less, more preferably about 2.5 e-10 orless, and most preferably about 2.5e-11 or less. The reprolysin domainhas been assigned the PFAM Accession number PF01421.

The consensus sequence for a reprolysin domain was derived from a hiddenMarkov model (HMM) within Pfam (version 5.5) (PFAM Accession numberPF01421). An alignment of the 65552 protein with a consensus reprolysinamino acid sequence (SEQ ID NO:80) has a predicted bit score of 9.3, andan E-value of 2.5e-11. Preferably, the reprolysin domain has the abilityto cleave a protein or peptide in the presence of a metal (i.e., Zn⁺⁺).

A reprolysin domain of a 65552 protein can also include a neutral zincmetallopeptidases, zinc-binding region having the following signaturesequence: [GSTALIVN]-x(2)-H-E-[LIVMFYW]-{DEHRKP}-H-x-[LIVMFYWGSPQ] (SEQID NO:83). In this signature sequence pattern, each element in thepattern is separated by a dash (-); square [ ] brackets indicate theparticular residues that are accepted at that position; elaborate{}brackets indicate the residues that are not accepted at that position;x indicates any residue is accepted at that position; a whole number inparenthesis following an x indicates any amino acid repetition of aparticular element is accepted for that specified number of residuesi.e., x(4), and the standard IUPAC one-letter code for the amino acidsis used. The two H's are zinc ligands and E is the active site residue.

This signature sequence can be found from about residues 358 to 367 ofthe 65552 protein (SEQ ID NO:77). For example, the conserved histidinescan be found at about amino acid residues 361 and 365 of SEQ ID NO:77.These histidine residues are believed to interact with zinc ions.

In a preferred embodiment, 65552 polypeptide or protein has a reprolysindomain or a region which includes at least about 100 to 300, morepreferably about 150 to 250, 175 to 225, 205 to 220 amino acid residuesand has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% identitywith a reprolysin domain, e.g., the domain of human 65552 (e.g.,residues 218 to 427 of SEQ ID NO:77) (SEQ ID NO:80). Preferably a 65552polypeptide or protein contains the typical neutral zincmetallopeptidases, zinc binding region signature sequence describedabove. This sequence is located at about amino acid residues 357 to 367of SEQ ID NO:77.

A 65552 polypeptide can also include one or more peptidase M12Bpropeptide domains. As used herein, the term “peptidase M12B propeptidedomain” refers to a protein domain having an amino acid sequence ofabout 40 to 160 amino acid residues in length, and having an E value ofabout 4.3 e-3. The peptidase M12B propeptide domain has a length ofabout 40 to 160 amino acid residues, preferably between about 60 to 150amino acids residues in length, more preferably between about 80 to 130amino acid residues in length, and even more preferably between about100 to 120 amino acid residues in length, and has an E-value of about4.3e-4 or less, more preferably 4.3-5 or less, and most preferably about4.3e-6 or less.

The peptidase M12B propeptide domain of the 65552 polypeptide showshomology to the peptidase M12B propeptide domain corresponding to PFAMAccession PF06562 derived from a hidden Markov model (HMM) within Pfam(version 5.5) (PFAM Accession number PF01562) (SEQ ID NO:79). Analignment of the 65552 protein (residues 67-181 of SEQ ID NO:77) with aconsensus peptidase M12B propeptide amino acid sequence derived from ahidden Markov model as predicted by PFAM has a bit score of 25.7, and anE-value of 24.3e-6.

In a preferred embodiment, a 65552 polypeptide or protein has apeptidase M12B propeptide domain or a region which includes at leastabout 40-160 amino acids, preferably about 60-150 amino acids, morepreferably about 80-130 amino acids, even more preferably about 100-120amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%,or 100% identity with a peptidase M12B propeptide domain (e.g., thepeptidase M12B propeptide domains of human 65552 (e.g., residues 67-181of SEQ ID NO:77).

The peptidase M12B propeptide domain may be involved in maintaining theprotease in a latent form. The propeptide contains a sequence motifsimilar to the “cysteine switch” of matrixins. The function of thedomain can be explained by the cysteine switch model, in whichcoordination of Zn2+ in the active site of the catalytic domain by acysteine residue in the prodomain is critical for inhibition of theprotease.

A 65552 polypeptide can also include one or more thrombospondin type 1domains. As used herein, the term “thrombospondin type 1 domain” alsoreferred to herein as “TSP-1 domain” refers to a protein domain havingan amino acid sequence of about 10 to 100 amino acid residues in length,and an E-value of about 1.5e-4. Preferably, the TSP-1 domain is between10 to 100 amino acid residues in length, preferably between about 20 to80 amino acids residues in length, more preferably between about 40 to60 amino acid residues in length, and even more preferably between about45 to 55 amino acids in length, and has an E-value of about 1.5e-5 orless, more preferably about 1.5e-5 or less, and most preferably about1.5e-7 or less. The thrombospondin type 1 domain has been assigned thePFAM Accession number PF00090. An alignment of the 65552 protein, (aminoacid residues 520-570 of SEQ ID NO:77) with a consensus thrombospondintype 1 amino acid sequence (SEQ ID NO:81) derived from a hidden Markovmodel within Pfam (version 5.5) (PFAM Accession number PF00090) (SEQ IDNO:81) has a bit score of 38.5, and an E-value of 1.5e-07.

In a preferred embodiment, a 65552 polypeptide or protein has athrombospondin type 1 domain or a region which includes at least about10-100 amino acids, preferably about 20-80 amino acids, more preferablyabout 40-60 amino acids, even more preferably about 45-55 amino acidresidues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%identity with a thrombospondin type 1 domain, e.g., the thrombospondintype 1 domains of human 65552 (e.g., 520-570 of SEQ ID NO:77).

Preferably, the thrombospondin type 1 domain has the ability to suppresstumor growth by its ability to inhibit neovascularization (angiogenesis)and proliferation. Preferably as well, the thrombospondin type 1 domainhas the ability to modulate cell-cell interaction (i.e., modulatingendothelial cell growth, adhesion, and motility and apoptosis).

To identify the presence of a reprolysin domain profile, a peptidaseM12B propeptide domain profile, and a thrombospondin type 1 domainprofile in a 65552 polypeptide, the amino acid sequence of the proteinis searched against a database of HMMs (e.g., the Pfam database, release2.1) using the default parameters. For example, the hmmsf program, whichis available as part of the HMMER package of search programs, is afamily specific default program for PF01421, PF01562, and PF00090 andscore of 15 is the default threshold score for determining a hit. Usingthe hmmsf program, the following domains were identified: a reprolysindomain profile was identified in the amino acid sequence of SEQ ID NO:77(e.g., amino acids 218-427 of SEQ ID NO:77 (SEQ ID NO:80)); a peptidaseM12B propeptide was identified in the amino acid sequence of SEQ IDNO:77 (e.g., amino acids 67-181 of SEQ ID NO:77 (SEQ ID NO:79)), and athrombospondin type 1 domain profile was identified in the amino acidsequence of SEQ ID NO:77 (e.g., amino acids 520-570 of SEQ ID NO:77 (SEQID NO:81)). Accordingly, a 65552 protein, or a domain thereof having atleast about 60-70%, more preferably about 70-80%, or still morepreferably about 80-90% homology with the reprolysin domain profile, andthe peptidase M12B propeptide domain profile, and the thrombospondintype 1 domain profile of human 65552 are within the scope of theinvention.

For further identification of polyptides, and demonstration of sharedcharacteristics with other metalloproteases, the 65552 polypeptidemolecule is aligned to that of other metaloproteases. An alignment ofhuman 65552 protein (SEQ ID NO:77) with a murine metalloproteinase(Swissprot Accession #: O54768: SEQ ID NO:82) demonstrates that thesesequences are 43.4% identical, and have a global alignment score of2516.

The human 65552 polypeptide can also contains a leucine zipper pattern,or regions homologous with a leucine zipper pattern.

Metalloproteases, fragments or variants thereof can have a leucinezipper motif or regions homologous with a leucine zipper motif. Leucinezippers typically contain a repeat of leucine positioned every sevenamino acids (L-x(6)-L-x(6)-L-x(6)-L) (SEQ ID NO:85), over a distance ofeight helical turns. The segments containing these periodic arrays ofleucines appear to exist in an alpha-helical conformation in whichleucine side chains extending from one alpha-helix interact with thosefrom a similar alpha helix of a second polypeptide, facilitatingdimerization. These interactions are frequently required for theactivity of the protein complex, e.g., transcriptional activation of anucleic acid via binding to a gene regulatory sequence and subsequentformation of a transcription initiation complex. Leucine zipperstherefore mediate protein-protein interactions in vivo and inparticular, interactions between multi-subunit transcription factors(homodimers, heterodimers, etc.).

Thus, in another embodiment, a 65552 metalloprotease or fragment orvariant can have one or more activities of a leucine zipper motif, suchas binding to another polypeptide that has a leucine zipper, forexample, forming a dimer with a 65552 metalloprotease or fragment orvariant containing a leucine zipper. The presence of a leucine zippermotif indicates that 65552 metalloprotease can participate in differentpathways due to an ability to interact with different proteins via theleucine zipper motif. For example, the leucine zipper motif can allow65552 metalloprotease binding to a protein substrate which 65552 maythen cleave. Thus, the leucine zipper motif modulates or is involved inone or more activities or functions of 65552 metalloprotease through itsability to confer binding of 65552 metalloprotease to a target moleculeor binding partner. The term “leucine zipper activity,” when used inreference to a protein, means a protein having one or more activitiesassociated with leucine-zipper function as described herein or otherwiseknown in the art.

65552 proteins of the invention can have signal sequences. As usedherein, a “signal sequence” includes a peptide of at least about 15 or20 amino acid residues in length which occurs at the N-terminus ofsecretory and membrane-bound proteins and which contains at least about70% hydrophobic amino acid residues such as alanine, leucine,isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine. Ina preferred embodiment, a signal sequence contains at least about 10 to40 amino acid residues, preferably about 15-30 amino acid residues, morepreferably about 17 amino acid residues, and has at least about 60-80%,more preferably 65-75%, and more preferably at least about 70%hydrophobic residues. A signal sequence serves to direct a proteincontaining such a sequence to a lipid bilayer. Thus, in one embodiment,a 65552 protein contains a signal sequence at about amino acids 1 to 17of SEQ ID NO:77. The signal sequence is cleaved during processing of themature protein.

In one embodiment, a 65552 protein exists in a mature form which doesnot include a signal sequence. In this embodiment, the 65552 protein canhave a length of about 934 (e.g., 927, 928, 929, 930, 931, 932, 933,934, 935, 936, or 937) amino acid residues, corresponding to a proteinhaving an amino terminus at about residue 18 (e.g., at residues 15, 16,17, 18, 19 or 20) and having a carboxyl terminus at about residue 950 ofSEQ ID NO:77. In this embodiment, the protein is preferably notmembrane-bound, and is also preferably extracellular.

The human 65552 protein has four predicted N-glycosylation sites (PfamAccession number PS00001) at about amino acid residues 141-144, 591-594,623-626 and 679-682 of SEQ ID NO:77; two glycosaminoglycan attachmentsites (PFAM Accession Number PS00002) at about amino acid residues417-420 and 760-763; two cAMP and cGMP-dependent protein kinasephosphorylation sites (PFAM Accession Number PS00004) at about aminoacid residues 204-207 and 683-686 of SEQ ID NO:77; fourteen predictedprotein kinase C phosphorylation sites (PFAM Accession Number PS00005)at about amino acid residues 171-173, 203-205, 207-209, 288-290,303-305, 471-473, 575-577, 578-580, 594-596, 617-619, 611-613, 665-667,681-683, and 917-919 of SEQ ID NO:77; fourteen predicted casein kinaseII phosphorylation sites (PFAM Accession Number PS00006) located atabout amino acid residues 19-22, 317-320, 325-328, 337-340, 346-349,359-362, 432-435, 492-495, 578-581, 611-614, 707-710, 730-733, 744-747,and 764-767 SEQ ID NO:77; fifteen predicted N-myristoylation sites (PFAMAccession Number PS00008) at about amino acid residues 90-95, 105-110,121-126, 164-169, 175-180, 323-328, 352-357, 439-444, 476-481, 490-495,535-540, 551-556, 658-663, 673-678, and 761-766 of SEQ ID NO:77; onepredicted amidation site (PFAM Accession Number PS00009) at about aminoacid residues 36-39 of SEQ ID NO:77; one predicted leucine zipperpattern (PFAM Accession Number PS00029) at about amino acid residues238-259 of SEQ ID NO:77; one zinc binding signature (PFAM AccessionNumber PS00142) at about amino acid residues 358-367 of SEQ ID NO:77;and one P-II protein urydylation site at about amino acid residues129-134 of SEQ ID NO:77.

Information regarding PFAM identifiers, PS prefix and PF prefix domainidentification numbers can be found at Sonnhammer et al. (1997) Protein28:405-420.

In one embodiment of the invention, a 65552 polypeptide includes atleast one reprolysin domain. In another embodiment, the 65552polypeptide comprises at least one reprolysin domain and at least onepeptidase M12B propeptide domain. In still another embodiment, the 65552polypeptide contains at least one reprolysin domain, at least onepeptidase M12B propeptide domain, and at least one thrombospondin type-1domain.

The 65552 molecules of the present invention can further include one ormore of the N-glycosylation, glycosaminoglycan attachment, cAMP andcGMP-dependent protein kinase phosphorylation, protein kinase Cphosphorylation, casein kinase II phosphorylation, N-myristoylation,amidation, leucine zipper, zinc binding signature, and P-II proteinurydylation sites sites described herein.

Because the 65552 polypeptides of the invention can modulate65552-mediated activities, they can be used to develop novel diagnosticand therapeutic agents for 65552-mediated or related disorders, asdescribed herein.

As used herein, a “65552 activity,” “biological activity of 65552,” or“functional activity of 65552,” refers to an activity exerted (directlyor indirectly) by a 65552 protein, polypeptide or nucleic acid moleculeon, for example, a 65552-responsive cell or on a 65552 substrate (e.g.,a protein substrate) as determined in vivo or in vitro. In oneembodiment, a 65552 activity is a direct activity, such as associationwith a 65552 target molecule. A “target molecule” or “binding partner”of a 65552 protein is a molecule with which the 65552 protein binds orinteracts in nature. In an exemplary embodiment, such a target moleculeis a 65552 receptor. A 65552 activity can also be an indirect activity,such as a cellular signaling activity mediated by interaction of the65552 protein with a 65552 receptor.

The 65552 molecules of the present invention can have similar biologicalactivities as other MMP family members. For example, the 65552 proteinsof the present invention can modulate (directly or indirectly) any oneor more of the following activities: 1) smooth muscle cell function(i.e., the muscular component of visceral structures (i.e., bloodvessels, the gastrointestinal tract, the uterus, or the urinarybladder)); 2) function of muscular arterial cells. (i.e., cells ofradial arteries, (i.e., cells of the tunica intima or the tunicaadventicia; 3) catalyzed cleavage of covalent bonds within or betweenamino acid residues in, for example, ECM, cell-surface, andextracellular proteins; 4) degradation of ECM; 5) angiogenesis; 6)neurite growth; 7 tumor cell invasion or metastasis; 8) the ability tomodulate tissue or organ integrity; 9) wound healing; 10) endometrialcycling; 11) hair follicle cycling; 12) bone remodeling; 13) ovulation;14 embryonic development; or 15) apoptosis.

Other activities, as described herein, include the ability to modulatefunction, survival, morphology, proliferation and/or differentiation ofcells of tissues in which 65552 molecules are expressed. Thus, the 65552molecules can act as novel diagnostic targets and therapeutic agents forcontrolling disorders involving aberrant activities of these cells.

As used herein, the term “metalloprotease activity,” or “proteaseactivity” when used in reference to a protein, means a protein havingthe ability to cleave a protein substrate by hydrolysis of an amidebond. Typically, the ability to cleave a protein substrate depends uponthe presence of a metal ion, such as zinc. Thus, a 65552metalloproteinase or fragment or variant having metalloproteinaseactivity is capable of cleaving one or more protein substrates in thepresence of a metal, e.g., zinc. Thus, a 65552 metalloproteinase orfragment or variant having metalloproteinase activity is capable ofcleaving one or more protein substrates in the presence of zinc.

Activity assays for the metalloproteinase family members, such as 65552polypeptides, involve any of the known metalloproteinase, reprolysin, orthrombospondin-like activity or functions, as well asactivities/functions that may not typically be found in othermetalloproteinases. These assays include, but are not limited to: 1)binding extracellular matrix; 2) binding collagen or gelatin; 3) bindingintegrin; 4) binding zinc or other metals; 5) binding a2-macroglobulin;6) cleaving specific peptide substrates to produce fragments, affectingcell adhesion; 7) binding heparin or other sulfated glycosaminoglycan,such as heparan sulfate; 8) modulation of vascularization or vascularendothelial growth; 9) breaking down cartilage; 10) induction ofapoptosis of endothelial cells; 11) suppressing tumor growth; 12)modulating angiogenesis; 13) affecting cellular chemotaxis; 14)affecting cell-cell adhesion or cell-matrix interaction; 15) bindingintegrin; 16) and any of the other biological or functional propertiesof these proteins, including, but not limited to, those disclosedherein, and in the references cited herein. Further, assays can relateto changes in the protein, per se, and on the effects of these changes,for example, cleavage of the substrate, activation of the proteinfollowing cleavage, etc. Such assays are described in Tang et al. (1999)FEBS Letters 445:223-225 (for example, induction by interleukin I invitro and by intravenous administration of lipopolysaccharide in vivo,as well as effects on cell adhesion, motility, and growth); Abbaszade etal., (J Biol. Chem. 2000 August 18; 275(33):25791-7). (for example,products resulting from cleavage at the Glu-Ala site in cartilageexplants and chondrocyte cultures treated with interleukin I andretinoic acid, determination of aggrecan cleaving activity with andwithout hydroxamate inhibitors); (Kuno and Matsushima (1998) J Biol Chem273: 13912-13917) (binding to the extracellular matrix, binding tosulfated glycosaminoglycans, binding to heparan sulfate); Kuno et al. J(1999) Biol. Chem. June 25; 274(26):18821-6. (protease trapping ofa2-macroglobulin, furin processing); Tortorella et al. (1999) Science.284(5420):1664-6. (detection of aggrecan fragments, especially byneoepitope antibodies, inhibition of cleavage by ADAM-TS inhibitors,inhibition of pro-MMP activation); Vasquez et al., J Appl Physiol.(1998) October; 85(4):1421-8. (suppression of fibroblast growthfactor-2-induced vascularization in the cornea pocket assay andinhibition of vascular endothelial growth factor-induced angiogenesis inthe chorioallantoic membrane assay, inhibition of endothelial cellproliferation, competitive inhibition with endostatin, proliferation ofhuman dermal endothelial cells, use of the antiangiogenic region of theTSP-1 motif as bait); (Kuno et al. (1997) J Biol Chem 272: 556-562);Wolfsberg et al., Dev Biol. 1995 May; 169(1):378-83; Guilpin et al.(1988) J. Biol. Chem. 273:157-166 (α2-macroglobulin trapping, cleavageof prodomain at the furin site to generate active metalloproteinase);Rosendahl et al., (1997)(J. Biol. Chem. 272:24588-24593)) (TNF αprocessing). Recombinant assay systems include, but are not limited to,those described in Abbaszade et al., supra; Kuno et al. (1998), supra;Kuno et al. (1999), supra; Tortorella et al., supra; Vasquez et al.,supra, and Kuno et al. (1997), supra.

As used herein, the term “TSP activity” or “TSP function,” when used inreference to a protein, means a protein that has one or more activitiesassociated with a TSP e.g., a TSP-1 domain as described herein orotherwise known in the art. For example, TSP domains are involved incell adhesion, migration, proliferation, outgrowth or angiogenesis.Thus, a 65552 metalloproteinase or fragment or variant having a TSPactivity can mediate or modulate cell-cell adhesion (e.g., due to thepresence of 65552 metalloproteinase in extracellular matrix),motility/migration, proliferation, outgrowth or angiogenesis, forexample. TSP domains also have been implicated in inflammatoryconditions and, therefore, a 65552 metalloproteinase or fragment orvariant with a TSP domain can participate in a pathway that affects aninflammatory response.

The 65552 metalloproteinase molecules find use in modulating 65552metalloproteinase function, activity, or expression, or relatedresponses to metalloproteinase function, activity or expression. As usedherein, the term “modulate” or grammatical variations thereof meansincreasing or decreasing an activity, function, signal or response. Thatis, the 65552 metalloproteinase molecules of the invention affect thetargeted activity in either a positive or negative fashion (e.g.,increase or decrease activity, function, or signal). Accordingly, the65552 molecules can act as novel diagnostic targets and therapeuticagents for controlling metalloproteinase disorders.

Thus, 65552 molecules described herein can act as novel diagnostictargets and therapeutic agents for prognosticating, modulating,diagnosing, preventing, inhibiting, alleviating, or treatingmetalloproteinase-associated disorders.

As used herein, a “metalloproteinase-associated disorder”(MMP-associated disorder) includes a disorder, disease or conditionwhich is characterized by a misregulation of a metalloproteinasemediated activity or by an abnormal metalloproteinase mediated activity.Metalloproteinase-associated disorders can detrimentally affect cellproliferation, cell adhesion, cell motility and migration, tissuestructural integrity (e.g., connective tissue formation andmaintenance), inflammatory response, erythroid cell activity, geneexpression, or angiogenesis and vascularization, among others. Thus,examples of metalloproteinase associated disorders in which the 65552molecules of the invention can be directly or indirectly involvedinclude cellular proliferative and/or differentiative disorders;disorders associated with undesirable or deficientvascularization/angiogenesis; disorders associated with undesirable ordeficient cell adhesion, motility or migration, including, e.g.,metastasis; disorders associated with undesirable or deficient tissuestructural integrity; disorders associated with undesirableextracellular matrix accumulation, e.g., characterized by fibrosis or ascar; inflammatory disorders, erythroid cell associated disorders; geneexpression disorders; and bleeding/clotting disorders.

The 65552 metalloproteinase molecules also find use in diagnosis ofdisorders involving an increase or decrease in 65552 metalloproteinaseexpression relative to normal expression, such as a proliferativedisorder, a differentiative disorder (e.g., cancer), an immune disorder,an erythroid cell-associated disorder; a motility disorder, a vasculardisorder, a bleeding or clotting disorder, or a developmental disorder.Thus, where expression or activity of 65552 metalloproteinase is greateror less than normal, this may indicate the presence of or apredisposition towards a 65552 metalloproteinase disorder. The presenceof 65552 metalloproteinase RNA or protein, e.g., by hybridization of a65552 specific probe or with a 65552 specific antibody, can be used toidentify the amount of 65552 present in a particular cell or tissue, orother biological sample. 65552 activity (protease activity assays,adhesion assays, binding assays, motility/migration assays,vascularization assays, etc.) can be assessed using the varioustechniques described herein or otherwise known in the art. Thus, inanother embodiment, the invention provides methods and compositions fordetection of 65552 metalloproteinase in tissues that normally or do notnormally express 65552 metalloproteinase.

The 65552 molecules and modulators thereof can act as novel therapeuticagents for controlling one or more of cellular proliferative and/ordifferentiative disorders, cardiovascular disorders, blood vesseldisorders as described herein.

Tissue Distribution of 65552 mRNA

TaqMan analysis indicates the highest levels of 65552 expression are inaortic smooth muscle cells, and muscular artery cells. 65552 is alsoexpressed in adipose tissue, human umbilical vein epithelial cells,diseased aorta cells, and cells of the vein

Human 65577

The present invention is based, in part, on the discovery of a novelgene, referenced to herein as 65577, which encodes a matrixmetalloprotease (also referred to herein as a matrix metalloproteinase,or an MMP), which is a member of the reprolysin (M12B) subfamily of theM12 family of metalloproteinase.

The human 65577 cDNA sequence which is approximately 3445 nucleotideresidues long including un-translated regions, contains a predictedmethionine-initiated coding sequence of about 3243 nucleotide residues,excluding termination codon (i.e., nucleotide residues 83-3325 of SEQ IDNO:86; 1-3243 of SEQ ID NO:88). The coding sequence encodes a 1081 aminoacid protein having the amino acid sequence SEQ ID NO:87.

The 65577 protein contains a significant number of structuralcharacteristics in common with members of the reprolysin (M12B)subfamily of MMPs. Like other members of this subfamily of MMPs, the65577 proteins of the invention can include a peptidase M12B-propeptidedomain having a sequence motif similar to the cysteine switch motif ofthe matrixins, and a reprolysin-like domain containing a zinc bindingsite (e.g., HEXXH; SEQ ID NO:84). Unlike the other members of thereprolysin (M12B) subfamily of MMPs, the 65577 proteins of the inventiondo not include an EGF-like domain, or a disintigrin domain.

The term “family” when referring to the protein and nucleic acidmolecules of the invention means two or more proteins or nucleic acidmolecules having a common structural domain or motif and havingsufficient amino acid or nucleotide sequence homology as defined herein.Such family members can be naturally or non-naturally occurring and canbe from either the same or different species. For example, a family cancontain a first protein of human origin as well as other distinctproteins of human origin, or alternatively, can contain homologues ofnon-human origin, e.g., matrix metalloprotease proteins for any speciesdescribed in the art (e.g., Steiner et al. (1995) Mol. Microbiol.16:825-834, and references cited therein). Members of a family can alsohave common functional characteristics.

Metalloproteases are a group of highly diverse, widely distributedproteolytic enzymes that depend on bound Ca²⁺ or Zn²⁺ for activity.Certain metalloproteases can readily utilize Mn²⁺ and Mg²⁺ as well.About 30 families of metalloproteases are recognized, about half ofwhich comprise enzymes containing the HEXXH motif (Rawlings et al.(1995) Meth Enzymol 248:183-228) (SEQ ID NO:84). The most thoroughlycharacterized of the metalloproteases is thermolysin, a member of the M4metalloprotease family.

Another metalloprotease family, the M12 family, contains the reprolysin(M12B) subfamily, which contains the snake venom metalloproteases andadamalysins family. The known members of the reprolysin subfamily mostlylack essential peptidase active sites, but typically contain a putativezinc-chelating sequence HELGHNLGMKH (SEQ ID NO:93), characteristic forthe reprolysin family of zinc-metalloproteinases. The reprolysin familyalso contains six cysteine residues in standard positions for this groupof proteins suggesting disulfide bonding (Leonardi A J et al. (1999)Chromatogrphy; 852 (1):237-43). These include BRCA1, a human breastcancer-associated protein, and mammalian fertilin.

In addition, there is a propeptide region for members of the peptidasefamily M12B. The propeptide contains a sequence motif similar to the“cysteine switch” of the matrixins. Matrix metalloproteinases” areexpressed as latent proenzymes that are activated by proteolyticcleavage that triggers a conformational change in the propeptide(cysteine switch) model, in which coordination of Zn²⁺ in the activesite of the catalytic domain by a cysteine residue in the prodomain iscritical for inhibition of the protease. (Overgaard et al. (1999) JBiol. Chem, 7:274(19):13427-33).

In general, the biological functions of metalloproteases include proteinmaturation, degradation of proteins, such as extracellular matrixproteins, tumor growth, metastasis and angiogenesis, among others. Thus,metalloproteases are likely to play important roles in a wide range ofdiseases including, but not limited to, cancer, arthritis, Alzheimer'sdisease, and a variety of inflammatory conditions other normal andpathological processes in which matrix metalloproteinase-catalyzedchanges in extracellular matrix protein structures have been implicatedare described, for example in Nagase et al. (1999) J. Biol. Chem.274:21491-21494. Accordingly, metalloproteases are an important targetfor drug action and development. Therefore, it is valuable to the fieldof pharmaceutical development to identify and characterize previouslyunknown metalloproteases.

65577 proteins of the invention can have signal sequences. As usedherein, a “signal sequence” includes a peptide of at least about 40 or50 amino acid residues in length which occurs at the N-terminus ofsecretory and membrane-bound proteins and which contains at least about70% hydrophobic amino acid residues such as alanine, leucine,isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine. Ina preferred embodiment, a signal sequence contains at least about 20 to60 amino acid residues, preferably about 30-50 amino acid residues, morepreferably about 47 amino acid residues, and has at least about 60-80%,more preferably 65-75%, and more preferably at least about 70%hydrophobic residues. A signal sequence serves to direct a proteincontaining such a sequence to a lipid bilayer. Thus, in one embodiment,a 65577 protein contains a signal sequence at about amino acids 1 to 47of SEQ ID NO:87. The signal sequence is cleaved during processing of themature protein.

In one embodiment, a 65577 protein exists in a mature form which doesnot include a signal sequence (e.g., in a form which does not includeresidues 1 to about 47 of SEQ ID NO:87). In this embodiment, the 65577protein can have a length of about 1035 (e.g., 1027, 1028, 1029, 1030,1031, 1032, 1033, 1034, 1035, 1036, 1037) amino acid residues,corresponding to a protein having an amino terminus at about residue 48and having a carboxyl terminus at about residue 1081 of SEQ ID NO:87.

In another embodiment, rather than a signal sequence at about residues 1to 47 of SEQ ID NO:87, a 65577 protein may include at least onetransmembrane domain at about amino acid residues 31 to 47 of SEQ IDNO:87. As used herein, the term “transmembrane domain” includes an aminoacid sequence of about 5 amino acid residues in length that spans theplasma membrane. More preferably, a transmembrane domain includes aboutat least 10, 15, 20 or 22 amino acid residues and spans a membrane.Transmembrane domains are rich in hydrophobic residues, and typicallyhave an alpha-helical structure. In a preferred embodiment, at least,60%, 70%, 80%, 90%, 95%, 99% or 100% or more of the amino acids of atransmembrane domain are hydrophobic, e.g., leucines, isoleucines,tyrosines, or tryptophans. Transmembrane domains are described in, forexample, Zagotta W. N. et al. (1996) Annu. Rev. Neurosci. 19: 235-263,the contents of which are incorporated herein by reference. Thus, aminoacid residues 31 to 47, 153-169 and 331-347 of SEQ ID NO:87 canalternatively comprise transmembrane domains in a 65577 protein.

A human 65577 polypeptide can also include various other domains orregions. A 65577 polypeptide can also include a peptidase M12Bpropeptide domain. As used herein, the term “peptidase M12B propeptidedomain” refers to a protein domain having an amino acid sequence ofabout 40-160 amino acid residues in length, preferably between about60-150 amino acid residues, more preferably between about 80-130 aminoacid residues, and even more preferably between about 100-120 amino acidresidues, and a bit score of about 30 or greater, preferably 40 orgreater, and most preferably 50 or greater, and an E-value of about2.3e-10 or less, more preferably about 2.3e-11 or less, and mostpreferably about 2.3e-12 or less when aligned with a M12B peptidasepropeptide domain (SEQ ID NO:89) derived from a hidden Markov model(HMM) with PFAM (PFAM Accession No. PF01562. The M12B propeptide domainin 65577 has a bit score of 57.8, and an e-value of 2.3e-13 when alignedto this consensus sequence. An alignment of the 65577 protein withconsensus peptidase M12B propeptide amino acid sequence (SEQ ID NO:89)(PFAM Accession No. PF01562) derived from a hidden Markov model showsthat a peptidase M12B propeptide domain of 65577 appears at aboutresidues 109-234 of SEQ ID NO:87.

In a preferred embodiment, a 65577 polypeptide or protein has apeptidase M12B propeptide domain or a region which includes at leastabout 50-175 amino acids, preferably about 75-150 amino acids, morepreferably about 100-125 amino acids, even more preferably about 110-120amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%,or 100% identity with a peptidase M12B propeptide domain, e.g., thepeptidase M12B propeptide domain of human 65577 (e.g., residues 109-234of SEQ ID NO:87).

The peptidase M12B propeptide domain contains conserved cysteines, anyone of which can be involved in the “cysteine switch” mechanism ofaction of these family members. It is believed that metalloproteinasesexist in a latent form. Evidence suggests that this latency is theresult of formation of an intramolecular complex between the singlecysteine residue in its propeptide domain (referred to herein as thepeptidase M12B propeptide domain) and the essential zinc atom in thecatalytic domain (referred to herein as a reprolysin-like domain), acomplex that blocks the active site. Latent metalloproteinase in thepresence of matrix degrading enzyme (i.e. collagenase) can be activatedby multiple means, all of which effect the dissociation of the cysteineresidue from the complex. This is referred to as the “cysteine-switch”mechanism of activation. The reprolysin-like domain contains the typicalHEXXH motif (SEQ ID NO:84), characteristic of enzymes that cleavepeptides. The histidines are positioned close together and act as zincligands, that are required for catalysis. The propeptide domain thatcontains the critical cysteine residue, and the catalytic domain thatcontains the zinc-binding site are the only two domains common to all ofthe MMPs. The amino acid sequences surrounding both the criticalcysteine residue and a region of the protein chains containing two ofthe putative histidine zinc-binding ligands are highly conserved in allof the MMPs. (Van Wart et al., (1990) Proc Natl Acad Sci USA;87(14):5578-82).

To identify the presence of a peptidase M12B propeptide domain profilesin a 65577 protein, the amino acid sequence of the protein is searchedagainst a database of HMMs (e.g., the Pfam database, release 2.1) usingthe default parameters. For example, the hmmsf program, which isavailable as part of the HMMER package of search programs, is a familyspecific default program for MILPAT0063 and score of 15 is the defaultthreshold score for determining a hit.

For further characterization of 65577 molecule of the invention as amatrix metalloprotease, the peptide sequence is searched against adatabase of proteins. An alignment of amino acid residues 1-1081 ofhuman 65577 (SEQ ID NO:87) with amino acid residues 1-1235 of a humanmetalloproteinase (SEQ ID NO:91) (TrEMBL:095428) demonstrates that thesesequences are 19.0% identical using a BLOSUM 50 scoring matrix and gappenalties of −12/2.

A 65577 protein can also include a reprolysin-like domain, which is thecatalytic domain of the protein. As used herein, the term“reprolysin-like domain” refers to a protein domain having an amino acidsequence of about 100-300 amino acid residues in length, preferablyabout 150-250 amino acid residues, more preferably about 175-225 aminoacid residues, and even more preferably about 205-220 amino acidresidues, and has a bit score of −50 or greater, preferably −40 orgreater, and most preferably −30 or greater, and an E-value of about 1.8e-3 or less, more preferably about 1.8 e-4 or less, and most preferablyabout 1.8-5 or less when aligned with a consensus reprolysin amino acidsequence (SEQ ID NO:90) from a hidden Markov model (HMM) within PFAM(PFAM Accession No. PF01421). The reprolysin-like domain in 65577 aspredicted by PFAM has a bit score of −28.2, and and E value of 1.8e-6.An alignment of the 65577 protein with the consensus reprolysin aminoacid sequence (SEQ ID NO:90) within PFAM (PFAM Accession No. PF01421)shows that a reprolysin-like domain of 65577 appears at residues 295 to497 of SEQ ID NO:87.

The reprolysin-like domain typically includes the following consensussequence: [GSTALIVN]-x(2)—H-E-[LIVMFYW]-{DEHRKP}-H-x-[LIVMFYWGSPQ] (SEQID NO:92) (PROSITE Pattern PDOC00129).

In this consensus sequence pattern, each element in the pattern isseparated by a dash (-); square [ ] brackets indicate the particularresidues that are accepted at that position; x indicates any residue isaccepted at that position; a whole number in parenthesis following an xindicates any amino acid repetition of a particular element is acceptedfor that specified number of residues i.e. x(2); { } brackets indicatethat the particular amino acid in that position can be any except thoseenclosed in the bracket.

The 65577 polypeptide of the invention contains a reprolysin-likeconsensus sequence at amino acid residues 432 to 441 of SEQ ID NO:87which represents 90% of the reprolysin consensus pattern described inPROSITE pattern PDOC00129. The reprolysin consensus sequence of the65577 polypeptide differs at amino acid residue 437 of SEQ ID NO:87,wherein an “S” is substituted for any of the “LIVMFY or W” residuescharacteristic of this sequence. The two histidines in this consensussequence bind zinc and are part of the conserved HEXXH motif (SEQ IDNO:84). The HEXXH motif is located at about amino acid residues 435 to439 of SEQ ID NO:87.

In a preferred embodiment, a 65577 polypeptide or protein has areprolysin-like domain or a region which includes at least about 90-270amino acids, preferably about 135-225 amino acids, more preferably about155-200 amino acids, even more preferably about 175-185 amino acidresidues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%identity with a reprolysin domain, e.g., the reprolysin-like domain ofhuman 65577 (e.g., residues 295 to 497 of SEQ ID NO:87).

The reprolysin domain is characteristic of extracellularmetalloproteases, such as collagenase and stromelysin, which degrade theextracellular matrix. The members of this family are enzymes that cleavepeptides. These proteases require zinc for catalysis, ligands for whichcan be histidine residues. Members of this family containing thereprolysin domain are also known as adamalysins.

To identify the presence of a reprolysin-like domain profile in a 65577protein, the amino acid sequence of the protein is searched against adatabase of HMMs (e.g., the Pfam database, release 2.1) using thedefault parameters. For example, the hmmsf program, which is availableas part of the HMMER package of search programs, is a family specificdefault program for MILPAT0063 and score of 15 is the default thresholdscore for determining a hit.

The human 65577 protein has six predicted N-glycosylation sites (PfamAccession number PS00001) at about amino acid residues 151-154, 190-193,313-316, 744-747, 837-840, and 908-911 of SEQ ID NO:87; one cAMP andcGMP-dependent protein kinase phosphorylation sites (PFAM AccessionNumber PS00004) at about amino acid residues 86-89 of SEQ ID NO:87;fourteen predicted protein kinase C phosphorylation sites (PFAMAccession Number PS00005) at about amino acid residues 135-137, 171-173,220-222, 279-281, 289-291, 395-397, 453-455, 630-623, 697-699, 746-748,794-796, 870-872, 913-915 and 952-954 of SEQ ID NO:87; nine predictedcasein kinase II phosphorylation sites (PFAM Accession Number PS00006)located at about amino acid residues 65-68, 206-209, 389-392, 420-423,676-676, 816-819, 1020-1023, 1024-1027 and 1059-1062 of SEQ ID NO:87;one tyrosine kinase phosphorylation site (PFAM Accession Number PS00007)at about amino acid residues 263-270 of SEQ ID NO:87; sixteen predictedN-myristoylation sites (PFAM Accession Number PS00008) at about aminoacid residues 73-78, 131-136, 167-172, 219-224, 312-317, 331-336,353-358, 426-431, 562-567, 589-584, 738-743, 752-757, 865-870, 890-895,948-953 and 1041-1046 of SEQ ID NO:87; and three predicted amidationsites (PFAM Accession Number PS00009) at about amino acid residues83-86, 254-257 and 378-381 of SEQ ID NO:87.

General information regarding PFAM identifiers, PS prefix and PF prefixdomain identification numbers can be found at Sonnhammer et al. (1997)Protein 28:405-420.

In one embodiment of the invention, a 65577 polypeptide includes atleast one reprolysin-like domain. In another embodiment, the 65577polypeptide includes at least one reprolysin-like domain and at leastone peptidase M12B propeptide domain. In still another embodiment, the65577 polypeptide contains at least one reprolysin-like domain, at leastone peptidase M12B propeptide domain, and at least one transmembranedomain.

The 65577 molecules of the present invention can further include one ormore of the N-glycosylation, cAMP and cGMP-dependent protein kinasephosphorylation, protein kinase C phosphorylation, casein kinase IIphosphorylation, tyrosine kinase phosphorylation, N-myristoylation, andamidation sites described herein.

Because the 65577 polypeptides of the invention can modulate 65577activities, they can be used to develop novel diagnostic and therapeuticagents for 65577-mediated or related disorders, as described herein.

As used herein, a “65577 activity,” “biological activity of 65577,” or“functional activity of 65577,” refers to an activity exerted (directlyor indirectly) by a 65577 protein, polypeptide or nucleic acid moleculeon, for example, a 65577-responsive cell or on a 65577 substrate (e.g.,a protein substrate) as determined in vivo or in vitro. In oneembodiment, a 65577 activity is a direct activity, such as associationwith a 65577 target molecule. A “target molecule” or “binding partner”of a 65577 protein is a molecule with which the 65577 protein binds orinteracts in nature. In an exemplary embodiment, such a target moleculeis a 65577 receptor (e.g. an ECM protein). A 65577 activity can also bean indirect activity, such as a cellular signaling activity mediated byinteraction of the 65577 protein with a 65577 receptor.

The 65577 molecules of the present invention have similar biologicalactivities as other MMP family members. For example, the 65577 proteinsof the present invention can have (directly or indirectly) any one ormore of the following activities: (1) the ability to cleave or modulatethe degredation of proteins or peptides of the extracellular matrix incells of the cardiovascular system. Examples of such cells in which the65577 molecule can act include arterial cells (e.g., arterial smoothmuscle cells (e.g., coronary arterial smooth muscle cells)), and venouscells (e.g., venous smooth muscle cells); (2) the ability to cleave ormodulate the degradation of peptides in cells of the central nervoussystem (e.g., brain cortex, spinal cord (e.g., schwann cells, neuronalcells, and glial cells (e.g., astrocytes)); (3) the ability to catalyzeor modulate catalysis of cleavage of covalent bonds within or betweenamino acid residues, e.g., in ECM, cell-surface, and extracellularproteins; (4) the ability to degrade ECM; (5) the ability to modulateangiogenesis; (6) the ability to modulate neurite growth; (7) theability to modulate tumor cell invasion or metastasis; (8) the abilityto modulate tissue or organ integrity; (9) the ability to modulate woundhealing; (10) the ability to modulate endometrial cycling; (11) theability to modulate hair follicle cycling; (12) the ability to modulatebone remodeling; (13) the ability to modulate ovulation; (14) theability to modulate embryonic development; and (15) the ability tomodulate apoptosis.

Other activities of the 65577 molecules of the invention include theability to modulate function, survival, morphology, proliferation and/ordifferentiation of cells of tissues in which 65577 molecules areexpressed. Thus, the 65577 molecules can act as novel diagnostic targetsand therapeutic agents for controlling disorders involving aberrantactivities of these cells.

Still other activities of the 65577 molecules of the invention includethe ability to cleave a protein substrate by hydrolysis of an amidebond. Typically, this ability of the molecules of the invention tocleave a protein substrate depends upon the presence of a metal ion,such as zinc. Thus, a 65577 molecule or subsequence or variant havingmetalloproteinase activity is capable of cleaving one or more proteinsubstrates in the presence of a metal, e.g., zinc. Thus, a 65577metalloprotease or subsequence or variant can cleave one or more proteinsubstrates in the presence of zinc.

Activity assays for the metalloproteinase family members, such as 65577polypeptides, involve any of the known metalloproteinase, reprolysin, orpeptidase M12B propeptide activity or functions, as well asactivities/functions that may not typically be found in othermetalloproteinases. These assays include assays which test the abilityto modulate (directly or indirectly) any one or more of the followingMMP functions: (1) the ability to cleave or modulate the degredationproteins or peptides of the extracellular matrix in cells of thecardiovascular system. Examples of such cells in which the 65577molecule can act include arterial cells (e.g., arterial smooth musclecells (e.g., coronary arterial smooth muscle cells)), and venous cells(e.g., venous smooth muscle cells); (2) the ability to cleave ormodulate the degradation of peptides in cells of the central nervoussystem (e.g., brain cortex, spinal cord (e.g., schwann cells, neuronalcells, and glial cells (e.g. astrocytes)); (3) binding ECM; (4) bindingcollagen or gelatin; (5) binding integrin; (6) binding zinc or othermetals; (7) binding a2-macroglobulin; (8) cleaving specific peptidesubstrates to produce fragments, affecting cell adhesion; (9) bindingheparin or other sulfated glycosaminoglycan, such as heparan sulfate;(10) modulating vascularization or vascular endothelial growth; (11breaking down cartilage; (12) induceing apoptosis of endothelial cells;(13) suppressing tumor growth; (14) modulating angiogenesis; (15)affecting cellular chemotaxis; (16) affecting cell-cell adhesion orcell-matrix interaction; (17) and any of the other biological orfunctional properties of these proteins, including, but not limited to,those disclosed herein, and in the references cited herein. Further,assays can relate to changes in the protein, per se, and on the effectsof these changes, for example, cleavage of the substrate, activation ofthe protein following cleavage, etc. Such assays are described in Tanget al. (1999) FEBS Letters 445:223-225 (for example, induction byinterleukin I in vitro and by intravenous administration oflipopolysaccharide in vivo, as well as effects on cell adhesion,motility, and growth); (Abbaszade et al., (2000) J Biol Chem.18;275(33):25791-7) (for example, products resulting from cleavage atthe Glu-Ala site in cartilage explants and chondrocyte cultures treatedwith interleukin I and retinoic acid, determination of aggrecan cleavingactivity with and without hydroxamate inhibitors); (Kuno and Matsushima(1998) J Biol Chem 273: 13912-13917) (binding to the extracellularmatrix, binding to sulfated glycosaminoglycans, binding to heparansulfate); Kuno et al. (1999) J Biol Chem. June 25; 274(26):18821-6(protease trapping of a2-macroglobulin, furin processing); Tortorella etal. (1999) Science; 284(5420): 1664-6 (detection of aggrecan fragments,especially by neoepitope antibodies, inhibition of cleavage by ADAM-TSinhibitors, inhibition of pro-MMP activation); Vasquez et al., (1998) JAppl Physiol. October; 85(4):1421-8 (suppression of fibroblast growthfactor-2-induced vascularization in the cornea pocket assay andinhibition of vascular endothelial growth factor-induced angiogenesis inthe chorioallantoic membrane assay, inhibition of endothelial cellproliferation, competitive inhibition with endostatin, proliferation ofhuman dermal endothelial cells, use of the antiangiogenic region of theTSP-1 motif as bait); (Kuno et al. (1997) J Biol Chem 272: 556-562);Wolfsberg et al., Dev Biol. (1995) May; 169(1):378-83; Guilpin et al.(1988) J. Biol. Chem. 273:157-166 (α2-macroglobulin trapping, cleavageof prodomain at the furin site to generate active metalloproteinase);Rosendahl et al., (J. Biol. Chem. (1997) 272:24588-24593) (TNF αprocessing). Recombinant assay systems include, but are not limited to,those described in Abbaszade et al., supra; Kuno et al. (1998), supra;Kuno et al. (1999), supra; Tortorella et al., supra; Vasquez et al.,supra, and Kuno et al. (1997), supra.

The 65577 molecules find use in modulating the 65577 activitiesdescribed herein. As used herein, the term “modulate” or grammaticalvariations thereof means increasing or decreasing an activity, function,signal or response. That is, the 65577 molecules of the invention affectthe targeted activity in either a positive or negative fashion (e.g.,increase or decrease activity, function, or signal). Accordingly, the65577 molecules can act as novel diagnostic targets and therapeuticagents for controlling disorders involving such activities (e.g.,metalloproteinase activities).

Thus, 65577 molecules described herein can act as novel diagnostictargets and therapeutic agents for prognosticating, modulating,diagnosing, preventing, inhibiting, alleviating, or treatingmetalloproteinase-associated disorders

As used herein, a “metalloproteinase-associated disorder”(MMP-associated disorder) includes a disorder, disease or conditionwhich is characterized by a misregulation of a metalloproteinasemediated activity or by an abnormal metalloproteinase mediated activity.As used herein, a metalloproteinase mediated activity, is an activitymediated or involving a molecule which can cleave a protein or peptidesubstrate in the presence of a metal. (e.g., Ca²⁺, Zn²⁺, Mn²⁺, Mg²⁺).Metalloproteinase-associated disorders can detrimentally affect cellproliferation, cell adhesion, cell motility and migration, tissuestructural integrity (e.g., connective tissue formation andmaintenance), inflammatory response, erythroid cell activity, geneexpression; or angiogenesis and vascularization, among others. Thus,examples of metalloproteinase-associated disorders in which the 65577molecules of the invention can be directly or indirectly involvedinclude cellular proliferative and/or differentiative disorders;disorders associated with undesirable or deficientvascularization/angiogenesis; disorders associated with undesirable ordeficient cell adhesion, motility or migration, including, e.g.,metastasis; disorders associated with undesirable or deficient tissuestructural integrity; disorders associated with undesirableextracellular matrix accumulation, e.g., characterized by fibrosis or ascar; inflammatory disorders, erythroid cell associated disorders; geneexpression disorders; and bleeding/clotting disorders.

The 65577 molecules also find use in diagnosis of disorders involving anincrease or decrease in 65577 expression relative to normal expression,such as a proliferative disorder, a differentiative disorder (e.g.,cancer), an immune disorder, an erythroid cell-associated disorder; amotility disorder, a vascular disorder, a bleeding or clotting disorder,or a developmental disorder. Thus, where expression or activity of 65577is greater or less than normal, this may indicate the presence of or apredisposition towards a 65577 disorder. The presence of 65577 RNA orprotein, e.g., by hybridization of a 65577 specific probe or with a65577 specific antibody, can be used to identify the amount of 65577present in a particular cell or tissue, or other biological sample.65577 activity (protease activity assays, adhesion assays, bindingassays, motility/migration assays, vascularization assays, etc.) can beassessed using the various techniques described herein or otherwiseknown in the art. Thus, in another embodiment, the invention providesmethods and compositions for detection of 65577 in tissues that normallyor do not normally express 65577.

The 65577 molecules and modulators thereof can act as novel therapeuticagents for controlling one or more of cardiovascular disorders, orneurological disorders as described herein.

Tissue Distribution of 65577 mRNA

TaqMan analysis indicates the highest levels of 65577 expression are innormal artery cells, normal vein cells, aortic and coronary smoothmuscle cells, and human umbilical vein cells. Futhermore, the resultsindicate high levels of expression in brain cortex, and spinal cord.65577 is also expressed at lower levels in in glial cells, normal ovarycells, and skin cells.

Definitions

The 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 protein, fragments thereof, and derivatives and other variantsof the sequence in SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or87 thereof are collectively referred to as “polypeptides or proteins ofthe invention” or “27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 polypeptides or proteins”. Nucleic acidmolecules encoding such polypeptides or proteins are collectivelyreferred to as “nucleic acids of the invention” or “27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 nucleic acids.”

As used herein, the term “nucleic acid molecule” includes DNA molecules(e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) andanalogs of the DNA or RNA generated, e.g., by the use of nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “isolated or purified nucleic acid molecule” includes nucleicacid molecules which are separated from other nucleic acid moleculeswhich are present in the natural source of the nucleic acid. Forexample, with regards to genomic DNA, the term “isolated” includesnucleic acid molecules which are separated from the chromosome withwhich the genomic DNA is naturally associated. Preferably, an “isolated”nucleic acid is free of sequences which naturally flank the nucleic acid(i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology (1989) John Wiley & Sons, N.Y., 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and preferably 4) very high stringency hybridization conditions are0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are thepreferred conditions and the ones that should be used unless otherwisespecified.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577protein, preferably a mammalian 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein, and can furtherinclude non-coding regulatory sequences, and introns.

An “isolated” or “purified” polypeptide or protein is substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.In one embodiment, the language “substantially free” means preparationof 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 protein having less than about 30%, 20%, 10% and morepreferably 5% (by dry weight), of non-27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein (also referred toherein as a “contaminating protein”), or of chemical precursors ornon-27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 chemicals. When the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the protein preparation.The invention includes isolated or purified preparations of at least0.01, 0.1, 1.0, and 10 milligrams in dry weight.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 (e.g., the sequence of SEQ ID NO:1,3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75,76, 78, 86 or 88) without abolishing or more preferably, withoutsubstantially altering a biological activity, whereas an “essential”amino acid residue results in such a change. For example, amino acidresidues that are conserved among the polypeptides of the presentinvention, e.g., those present in the conserved domains, are predictedto be particularly unamenable to alteration.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO: 1, 3, 4, 6, 11, 13, 20, 22, 25, 27,29, 31, 35, 37, 38, 40, 41, 43, 73, 75, 76, 78, 86 or 88, the encodedprotein can be expressed recombinantly and the activity of the proteincan be determined.

As used herein, a “biologically active portion” of a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteinincludes a fragment of a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein which participates in aninteraction between a 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 molecule and a non-27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 molecule.Biologically active portions of a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein, e.g., the amino acidsequence shown in SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or87, which include fewer amino acids than the full length 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein,and exhibit at least one activity of a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 protein. A biologically active portion of a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein can be a polypeptide which is, for example, 10, 25, 50,100, 200 or more amino acids in length. Biologically active portions ofa 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 protein can be used as targets for developing agents whichmodulate a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 mediated activity.

Calculations of homology or sequence identity (the terms “homology” and“identity” are used interchangeably herein) between sequences areperformed as follows:

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (1970)J. Mol. Biol. 48:444-453 algorithm which has been incorporated into theGAP program in the GCG software package using either a Blossum 62 matrixor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set ofparameters (and the one that should be used if the practitioner isuncertain about what parameters should be applied to determine if amolecule is within a sequence identity or homology limitation of theinvention) are a Blossum 62 scoring matrix with a gap penalty of 12, agap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of Meyers and Miller ((1989) CABIOS,4:11-17) which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25:3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used.

Particular 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 polypeptides of the present invention have anamino acid sequence substantially identical to the amino acid sequenceof SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87. In thecontext of an amino acid sequence, the term “substantially identical” isused herein to refer to a first amino acid that contains a sufficient orminimum number of amino acid residues that are i) identical to, or ii)conservative substitutions of aligned amino acid residues in a secondamino acid sequence such that the first and second amino acid sequencescan have a common structural domain and/or common functional activity.For example, amino acid sequences that contain a common structuraldomain having at least about 60%, or 65% identity, likely 75% identity,more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87 aretermed substantially identical.

In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 60%, or 65%identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1, 3, 4, 6, 11, 13, 20,22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75, 76, 78, 86 or 88 aretermed substantially identical.

“Misexpression or aberrant expression”, as used herein, refers to anon-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

“Subject”, as used herein, can refer to a mammal, e.g., a human, or toan experimental or animal or disease model. The subject can also be anon-human animal, e.g., a horse, cow, goat, or other domestic animal.

A “purified preparation of cells”, as used herein, refers to, in thecase of plant or animal cells, an in vitro preparation of cells and notan entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

As used herein, cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver origin.

As used herein, the term “cancer” (also used interchangeably with theterms, “hyperproliferative” and “neoplastic”) refers to cells having thecapacity for autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. Cancerous diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, e.g., malignant tumor growth, or may becategorized as non-pathologic, i.e., a deviation from normal but notassociated with a disease state, e.g., cell proliferation associatedwith wound repair. The term is meant to include all types of cancerousgrowths or oncogenic processes, metastatic tissues or malignantlytransformed cells, tissues, or organs, irrespective of histopathologictype or stage of invasiveness. The term “cancer” includes malignanciesof the various organ systems, such as those affecting lung, breast,thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as wellas adenocarcinomas which include malignancies such as most coloncancers, renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus. The term “carcinoma” is art recognized andrefers to malignancies of epithelial or endocrine tissues includingrespiratory system carcinomas, gastrointestinal system carcinomas,genitourinary system carcinomas, testicular carcinomas, breastcarcinomas, prostatic carcinomas, endocrine system carcinomas, andmelanomas. Exemplary carcinomas include those forming from tissue of thecervix, lung, prostate, breast, head and neck, colon and ovary. The term“carcinoma” also includes carcinosarcomas, e.g., which include malignanttumors composed of carcinomatous and sarcomatous tissues. An“adenocarcinoma” refers to a carcinoma derived from glandular tissue orin which the tumor cells form recognizable glandular structures. Theterm “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, tumors such as bronchogeniccarcinoma, including paraneoplastic syndromes, bronchioloalveolarcarcinoma, neuroendocrine tumors, such as bronchial carcinoid,miscellaneous tumors, metastatic tumors, and pleural tumors, includingsolitary fibrous tumors (pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast diseaseincluding, e.g., epithelial hyperplasia, sclerosing adenosis, and smallduct papillomas; tumors, e.g., stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma,invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)carcinoma, tubular carcinoma, and invasive papillary carcinoma, andmiscellaneous malignant neoplasms. Disorders in the male breast include,but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disordersinvolving the colon include, but are not limited to, tumors of thecolon, such as non-neoplastic polyps, adenomas, familial syndromes,colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.

Examples of cancers or neoplastic conditions, in addition to the onesdescribed above, include, but are not limited to, a fibrosarcoma,myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer,rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer,uterine cancer, cancer of the head and neck, skin cancer, brain cancer,squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular cancer, small cell lung carcinoma, non-smallcell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma.

Proliferative disorders include hematopoietic neoplastic disorders. Asused herein, the term “hematopoietic neoplastic disorders” includesdiseases involving hyperplastic/neoplastic cells of hematopoieticorigin, e.g., arising from myeloid, lymphoid or erythroid lineages, orprecursor cells thereof. Preferably, the diseases arise from poorlydifferentiated acute leukemias, e.g., erythroblastic leukemia and acutemegakaryoblastic leukemia. Additional exemplary myeloid disordersinclude, but are not limited to, acute promyeloid leukemia (APML), acutemyelogenous leukemia (AML) and chronic myelogenous leukemia (CML)(reviewed in Vaickus (1991) Crit Rev. in Oncol./Hemotol. 11:267-97);lymphoid malignancies include, but are not limited to acutelymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineageALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).Additional forms of malignant lymphomas include, but are not limited tonon-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

As used herein, an “angiogenesis disorder” includes a disease ordisorder which affects or is caused by aberrant or deficientangiogenesis. Disorders involving angiogenesis include, but are notlimited to, aberrant or excess angiogenesis in tumors such ashemangiomas and Kaposi's sarcoma, von Hippel-Lindau disease, as well asthe angiogenesis associated with tumor growth; aberrant or excessangiogenesis in diseases such as a Castleman's disease or fibrodysplasiaossificans progressiva; aberrant or deficient angiogenesis associatedwith aging, complications of healing certain wounds and complications ofdiseases such as diabetes and rheumatoid arthritis; or aberrant ordeficient angiogenesis associated with hereditary hemorrhagictelangiectasia, autosomal dominant polycystic kidney disease,myelodysplastic syndrome or Klippel-Trenaunay-Weber syndrome.

As used herein, disorders of the breast include, but are not limited to,disorders of development; inflammations, including but not limited to,acute mastitis, periductal mastitis, periductal mastitis (recurrentsubareolar abscess, squamous metaplasia of lactiferous ducts), mammaryduct ectasia, fat necrosis, granulomatous mastitis, and pathologiesassociated with silicone breast implants; fibrocystic changes;proliferative breast disease including, but not limited to, epithelialhyperplasia, sclerosing adenosis, and small duct papillomas; tumorsincluding, but not limited to, stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma, nospecial type, invasive lobular carcinoma, medullary carcinoma, colloid(mucinous) carcinoma, tubular carcinoma, and invasive papillarycarcinoma, and miscellaneous malignant neoplasms. Disorders in the malebreast include, but are not limited to, gynecomastia and carcinoma.

As used herein, disorders involving the colon include, but are notlimited to, congenital anomalies, such as atresia and stenosis, Meckeldiverticulum, congenital aganglionic megacolon-Hirschsprung disease;enterocolitis, such as diarrhea and dysentery, infectious enterocolitis,including viral gastroenteritis, bacterial enterocolitis, necrotizingenterocolitis, antibiotic-associated colitis (pseudomembranous colitis),and collagenous and lymphocytic colitis, miscellaneous intestinalinflammatory disorders, including parasites and protozoa, acquiredimmunodeficiency syndrome, transplantation, drug-induced intestinalinjury, radiation enterocolitis, neutropenic colitis (typhlitis), anddiversion colitis; idiopathic inflammatory bowel disease, such as Crohndisease and ulcerative colitis; tumors of the colon, such asnon-neoplastic polyps, adenomas, familial syndromes, colorectalcarcinogenesis, colorectal carcinoma, and carcinoid tumors.

As used herein, disorders involving the kidney include, but are notlimited to, congenital anomalies including, but not limited to, cysticdiseases of the kidney, that include but are not limited to, cysticrenal dysplasia, autosomal dominant (adult) polycystic kidney disease,autosomal recessive (childhood) polycystic kidney disease, and cysticdiseases of renal medulla, which include, but are not limited to,medullary sponge kidney, and nephronophthisis-uremic medullary cysticdisease complex, acquired (dialysis-associated) cystic disease, such assimple cysts; glomerular diseases including pathologies of glomerularinjury that include, but are not limited to, in situ immune complexdeposition, that includes, but is not limited to, anti-GBM nephritis,Heymann nephritis, and antibodies against planted antigens, circulatingimmune complex nephritis, antibodies to glomerular cells, cell-mediatedimmunity in glomerulonephritis, activation of alternative complementpathway, epithelial cell injury, and pathologies involving mediators ofglomerular injury including cellular and soluble mediators, acuteglomerulonephritis, such as acute proliferative (poststreptococcal,postinfectious) glomerulonephritis, including but not limited to,poststreptococcal glomerulonephritis and nonstreptococcal acuteglomerulonephritis, rapidly progressive (crescentic) glomerulonephritis,nephrotic syndrome, membranous glomerulonephritis (membranousnephropathy), minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, IgAnephropathy (Berger disease), focal proliferative and necrotizingglomerulonephritis (focal glomerulonephritis), hereditary nephritis,including but not limited to, Alport syndrome and thin membrane disease(benign familial hematuria), chronic glomerulonephritis, glomerularlesions associated with systemic disease, including but not limited to,systemic lupus erythematosus, Henoch-Schonlein purpura, bacterialendocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary andimmunotactoid glomerulonephritis, and other systemic disorders; diseasesaffecting tubules and interstitium, including acute tubular necrosis andtubulointerstitial nephritis, including but not limited to,pyelonephritis and urinary tract infection, acute pyelonephritis,chronic pyelonephritis and reflux nephropathy, and tubulointerstitialnephritis induced by drugs and toxins, including but not limited to,acute drug-induced interstitial nephritis, analgesic abuse nephropathy,nephropathy associated with nonsteroidal anti-inflammatory drugs, andother tubulointerstitial diseases including, but not limited to, uratenephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma;diseases of blood vessels including benign nephrosclerosis, malignanthypertension and accelerated nephrosclerosis, renal artery stenosis, andthrombotic microangiopathies including, but not limited to, classic(childhood) hemolytic-uremic syndrome, adult hemolytic-uremicsyndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, andother vascular disorders including, but not limited to, atheroscleroticischemic renal disease, atheroembolic renal disease, sickle cell diseasenephropathy, diffuse cortical necrosis, and renal infarcts; urinarytract obstruction (obstructive uropathy); urolithiasis (renal calculi,stones); and tumors of the kidney including, but not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypernephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

Examples of disorders of the lung include, but are not limited to,congenital anomalies; atelectasis; diseases of vascular origin, such aspulmonary congestion and edema, including hemodynamic pulmonary edemaand edema caused by microvascular injury, adult respiratory distresssyndrome (diffuse alveolar damage), pulmonary embolism, hemorrhage, andinfarction, and pulmonary hypertension and vascular sclerosis; chronicobstructive pulmonary disease, such as emphysema, chronic bronchitis,bronchial asthma, and bronchiectasis; diffuse interstitial(infiltrative, restrictive) diseases, such as pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia(pulmonary infiltration with eosinophilia), Bronchiolitisobliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes,including Goodpasture syndrome, idiopathic pulmonary hemosiderosis andother hemorrhagic syndromes, pulmonary involvement in collagen vasculardisorders, and pulmonary alveolar proteinosis; complications oftherapies, such as drug-induced lung disease, radiation-induced lungdisease, and lung transplantation; tumors, such as bronchogeniccarcinoma, including paraneoplastic syndromes, bronchioloalveolarcarcinoma, neuroendocrine tumors, such as bronchial carcinoid,miscellaneous tumors, and metastatic tumors; pathologies of the pleura,including inflammatory pleural effusions, noninflammatory pleuraleffusions, pneumothorax, and pleural tumors, including solitary fibroustumors (pleural fibroma) and malignant mesothelioma.

As used herein, disorders involving the pancreas include those of theexocrine pancreas such as congenital anomalies, including but notlimited to, ectopic pancreas; pancreatitis, including but not limitedto, acute pancreatitis; cysts, including but not limited to,pseudocysts; tumors, including but not limited to, cystic tumors andcarcinoma of the pancreas; and disorders of the endocrine pancreas suchas, diabetes mellitus; islet cell tumors, including but not limited to,insulinomas, gastrinomas, and other rare islet cell tumors.

As used herein, disorders involving the ovary include, for example,polycystic ovarian disease, Stein-leventhal syndrome, Pseudomyxomaperitonei and stromal hyperthecosis; ovarian tumors such as, tumors ofcoelomic epithelium, serous tumors, mucinous tumors, endometeriodtumors, clear cell adenocarcinoma, cystadenofibroma, brenner tumor,surface epithelial tumors; germ cell tumors such as mature (benign)teratomas, monodermal teratomas, immature malignant teratomas,dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomaltumors such as, granulosa-theca cell tumors, thecoma-fibromas,androblastomas, hill cell tumors, and gonadoblastoma; and metastatictumors such as Krukenberg tumors.

As used herein, hormonal disorders and diseases include type I and typeII diabetes mellitus, pituitary disorders (e.g., growth disorders),thyroid disorders (e.g., hypothyroidism or hyperthyroidism), andreproductive or fertility disorders (e.g., disorders which affect theorgans of the reproductive system, e.g., the prostate gland, the uterus,or the vagina; disorders which involve an imbalance in the levels of areproductive hormone in a subject; disorders affecting the ability of asubject to reproduce; and disorders affecting secondary sexcharacteristic development, e.g., adrenal hyperplasia).

Aberrant expression and/or activity of the molecules of the inventioncan mediate disorders associated with bone metabolism. “Bone metabolism”refers to direct or indirect effects in the formation or degeneration ofbone structures, e.g., bone formation, bone resorption, etc., which canultimately affect the concentrations in serum of calcium and phosphate.This term also includes activities mediated by the molecules of theinvention in bone cells, e.g. osteoclasts and osteoblasts, that can inturn result in bone formation and degeneration. For example, moleculesof the invention can support different activities of bone resorbingosteoclasts such as the stimulation of differentiation of monocytes andmononuclear phagocytes into osteoclasts. Accordingly, molecules of theinvention that modulate the production of bone cells can influence boneformation and degeneration, and thus can be used to treat bonedisorders. Examples of such disorders include, but are not limited to,osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosacystica, renal osteodystrophy, osteosclerosis, anti-convulsanttreatment, osteopenia, fibrogenesis-imperfecta ossium, secondaryhyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis,obstructive jaundice, drug induced metabolism, medullary carcinoma,chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism,malabsorption syndrome, steatorrhea, tropical sprue, idiopathichypercalcemia and milk fever.

As used herein, “a prostate disorder” refers to an abnormal conditionoccurring in the male pelvic region characterized by, e.g., male sexualdysfunction and/or urinary symptoms. This disorder may be manifested inthe form of genitourinary inflammation (e.g., inflammation of smoothmuscle cells) as in several common diseases of the prostate includingprostatitis, benign prostatic hyperplasia and cancer, e.g.,adenocarcinoma or carcinoma, of the prostate.

Examples of immune, e.g., inflammatory, (e.g. respiratory inflammatory)disorders or diseases include, but are not limited to, autoimmunediseases (including, for example, diabetes mellitus, arthritis(including rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjögren's Syndrome, inflammatorybowel disease, e.g. Crohn's disease and ulcerative colitis, aphthousulcer, iritis, conjunctivitis, keratoconjunctivitis, asthma, allergicasthma, chronic obstructive pulmonary disease, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitisposterior, and interstitial lung fibrosis), graft-versus-host disease,cases of transplantation, and allergy such as, atopic allergy.

As used herein, disorders involving the heart, or “cardiovasculardisease” or a “cardiovascular disorder” includes a disease or disorderwhich affects the cardiovascular system, e.g., the heart, the bloodvessels, and/or the blood. A cardiovascular disorder can be caused by animbalance in arterial pressure, a malfunction of the heart, or anocclusion of a blood vessel, e.g., by a thrombus. A cardiovasculardisorder includes, but is not limited to disorders such asarteriosclerosis, atherosclerosis, cardiac hypertrophy, ischemiareperfusion injury, restenosis, arterial inflammation, vascular wallremodeling, ventricular remodeling, rapid ventricular pacing, coronarymicroembolism, tachycardia, bradycardia, pressure overload, aorticbending, coronary artery ligation, vascular heart disease, valvulardisease, including but not limited to, valvular degeneration caused bycalcification, rheumatic heart disease, endocarditis, or complicationsof artificial valves; atrial fibrillation, long-QT syndrome, congestiveheart failure, sinus node dysfunction, angina, heart failure,hypertension, atrial fibrillation, atrial flutter, pericardial disease,including but not limited to, pericardial effusion and pericarditis;cardiomyopathies, e.g., dilated cardiomyopathy or idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, ischemic disease, arrhythmia, sudden cardiac death, andcardiovascular developmental disorders (e.g., arteriovenousmalformations, arteriovenous fistulae, raynaud's syndrome, neurogenicthoracic outlet syndrome, causalgia/reflex sympathetic dystrophy,hemangioma, aneurysm, cavernous angioma, aortic valve stenosis, atrialseptal defects, atrioventricular canal, coarctation of the aorta,ebsteins anomaly, hypoplastic left heart syndrome, interruption of theaortic arch, mitral valve prolapse, ductus arteriosus, patent foramenovale, partial anomalous pulmonary venous return, pulmonary atresia withventricular septal defect, pulmonary atresia without ventricular septaldefect, persistance of the fetal circulation, pulmonary valve stenosis,single ventricle, total anomalous pulmonary venous return, transpositionof the great vessels, tricuspid atresia, truncus arteriosus, ventricularseptal defects). A cardiovascular disease or disorder also can includean endothelial cell disorder.

“Procedural vascular trauma” includes the effects ofsurgical/medical-mechanical interventions into mammalian vasculature,but does not include vascular trauma due to the organic vascularpathologies listed hereinabove, or to unintended traumas, such as due toan accident. Thus, procedural vascular traumas within the scope of thepresent treatment method include (1) organ grafting or transplantation,such as transplantation and grafting of heart, kidney, liver and thelike, e.g., involving vessel anastomosis; (2) vascular surgery, such ascoronary bypass surgery, biopsy, heart valve replacement, atheroectomy,thrombectomy, and the like; (3) transcatheter vascular therapies (TVT)including angioplasty, e.g., laser angioplasty and PTCA proceduresdiscussed hereinbelow, employing balloon catheters, or indwellingcatheters; (4) vascular grafting using natural or synthetic materials,such as in saphenous vein coronary bypass grafts, dacron and venousgrafts used for peripheral arterial reconstruction, etc.; (5) placementof a mechanical shunt, such as a PTFE hemodialysis shunt used forarteriovenous communications; and (6) placement of an intravascularstent, which may be metallic, plastic or a biodegradable polymer. SeeU.S. patent application Ser. No. 08/389,712, filed Feb. 15, 1995, whichis incorporated by reference herein. For a general discussion ofimplantable devices and biomaterials from which they can be formed, seeH. Kambic et al., “Biomaterials in Artificial Organs”, Chem. Eng. News,30 (Apr. 14, 1986), the disclosure of which is incorporated by referenceherein.

Small vessel disease includes, but is not limited to, vascularinsufficiency in the limbs, peripheral neuropathy and retinopathy, e.g.,diabetic retinopathy.

As used herein, disorders involving the brain include, but are notlimited to, disorders involving neurons, and disorders involving glia,such as astrocytes, oligodendrocytes, ependymal cells, and microglia;cerebral edema, raised intracranial pressure and herniation, andhydrocephalus; malformations and developmental diseases, such as neuraltube defects, forebrain anomalies, posterior fossa anomalies, andsyringomyelia and hydromyelia; perinatal brain injury; cerebrovasculardiseases, such as those related to hypoxia, ischemia, and infarction,including hypotension, hypoperfusion, and low-flow states—globalcerebral ischemia and focal cerebral ischemia—infarction fromobstruction of local blood supply, intracranial hemorrhage, includingintracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage andruptured berry aneurysms, and vascular malformations, hypertensivecerebrovascular disease, including lacunar infarcts, slit hemorrhages,and hypertensive encephalopathy; infections, such as acute meningitis,including acute pyogenic (bacterial) meningitis and acute aseptic(viral) meningitis, acute focal suppurative infections, including brainabscess, subdural empyema, and extradural abscess, chronic bacterialmeningoencephalitis, including tuberculosis and mycobacterioses,neurosyphilis, and neuroborreliosis (Lyme disease), viralmeningoencephalitis, including arthropod-borne (Arbo) viralencephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2,Varicella-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis,rabies, and human immunodeficiency virus 1, including HIV-1meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degenration,multiple system atrophy, including striatonigral degenration, Shy-Dragersyndrome, and olivopontocerebellar atrophy, and Huntington disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

As used herein, disorders involving blood vessels include, but are notlimited to, responses of vascular cell walls to injury, such asendothelial dysfunction and endothelial activation and intimalthickening; vascular diseases including, but not limited to, congenitalanomalies, such as arteriovenous fistula, atherosclerosis, andhypertensive vascular disease, such as hypertension; inflammatorydisease—the vasculitides, such as giant cell (temporal) arteritis,Takayasu arteritis, polyarteritis nodosa (classic), Kawasaki syndrome(mucocutaneous lymph node syndrome), microscopic polyanglitis(microscopic polyarteritis, hypersensitivity or leukocytoclasticanglitis), Wegener granulomatosis, thromboanglitis obliterans (Buergerdisease), vasculitis associated with other disorders, and infectiousarteritis; Raynaud disease; aneurysms and dissection, such as abdominalaortic aneurysms, syphilitic (luetic) aneurysms, and aortic dissection(dissecting hematoma); disorders of veins and lymphatics, such asvaricose veins, thrombophlebitis and phlebothrombosis, obstruction ofsuperior vena cava (superior vena cava syndrome), obstruction ofinferior vena cava (inferior vena cava syndrome), and lymphangitis andlymphedema; tumors, including benign tumors and tumor-like conditions,such as hemangioma, lymphangioma, glomus tumor (glomangioma), vascularectasias, and bacillary angiomatosis, and intermediate-grade (borderlinelow-grade malignant) tumors, such as Kaposi sarcoma andhemangloendothelioma, and malignant tumors, such as angiosarcoma andhemangiopericytoma; and pathology of therapeutic interventions invascular disease, such as balloon angioplasty and related techniques andvascular replacement, such as coronary artery bypass graft surgery.

As used herein, disorders involving the testis and epididymis include,but are not limited to, congenital anomalies such as cryptorchidism,regressive changes such as atrophy, inflammations such as nonspecificepididymitis and orchitis, granulomatous (autoimmune) orchitis, andspecific inflammations including, but not limited to, gonorrhea, mumps,tuberculosis, and syphilis, vascular disturbances including torsion,testicular tumors including germ cell tumors that include, but are notlimited to, seminoma, spermatocytic seminoma, embryonal carcinoma, yolksac tumor choriocarcinoma, teratoma, and mixed tumors, tumore of sexcord-gonadal stroma including, but not limited to, Leydig (interstitial)cell tumors and sertoli cell tumors (androblastoma), and testicularlymphoma, and miscellaneous lesions of tunica vaginalis.

As used herein, skeletal muscle disorders include, but are not limitedto, muscular dystrophy (e.g., Duchenne muscular dystrophy, Beckermuscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdlemuscular dystrophy, facioscapulohumeral muscular dystrophy, myotonicdystrophy, oculopharyngeal muscular dystrophy, distal musculardystrophy, and congenital muscular dystrophy), motor neuron diseases(e.g., amyotrophic lateral sclerosis, infantile progressive spinalmuscular atrophy, intermediate spinal muscular atrophy, spinal bulbarmuscular atrophy, and adult spinal muscular atrophy), myopathies (e.g.,inflammatory myopathies (e.g., dermatomyositis and polymyositis),myotonia congenita, paramyotonia congenita, central core disease,nemaline myopathy, myotubular myopathy, and periodic paralysis), tumorssuch as rhabdomyosarcoma, and metabolic diseases of muscle (e.g.,phosphorylase deficiency, acid maltase deficiency, phosphofructokinasedeficiency, debrancher enzyme deficiency, mitochondrial myopathy,carnitine deficiency, carnitine palmityl transferase deficiency,phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency,lactate dehydrogenase deficiency, and myoadenylate deaminasedeficiency).

As used herein, an “endothelial cell disorder” includes a disordercharacterized by aberrant, unregulated, or unwanted endothelial cellactivity, e.g., proliferation, migration, angiogenesis, orvascularization; or aberrant expression of cell surface adhesionmolecules or genes associated with angiogenesis, e.g., TIE-2, FLT andFLK. Endothelial cell disorders include tumorigenesis, tumor metastasis,psoriasis, diabetic retinopathy, endometriosis, Grave's disease,ischemic disease (e.g., atherosclerosis), and chronic inflammatorydiseases (e.g., rheumatoid arthritis).

Disorders which can be treated or diagnosed by methods described hereininclude, but are not limited to, disorders associated with anaccumulation in the liver of fibrous tissue, such as that resulting froman imbalance between production and degradation of the extracellularmatrix accompanied by the collapse and condensation of preexistingfibers. The methods described herein can be used to diagnose or treathepatocellular necrosis or injury induced by a wide variety of agentsincluding processes which disturb homeostasis, such as an inflammatoryprocess, tissue damage resulting from toxic injury or altered hepaticblood flow, and infections (e.g., bacterial, viral and parasitic). Forexample, the methods can be used for the early detection of hepaticinjury, such as portal hypertension or hepatic fibrosis. In addition,the methods can be employed to detect liver fibrosis attributed toinborn errors of metabolism, for example, fibrosis resulting from astorage disorder such as Gaucher's disease (lipid abnormalities) or aglycogen storage disease, A1-antitrypsin deficiency; a disordermediating the accumulation (e.g., storage) of an exogenous substance,for example, hemochromatosis (iron-overload syndrome) and copper storagediseases (Wilson's disease), disorders resulting in the accumulation ofa toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) andperoxisomal disorders (e.g., Zellweger syndrome). Additionally, themethods described herein can be used for the early detection andtreatment of liver injury associated with the administration of variouschemicals or drugs, such as for example, methotrexate, isonizaid,oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, orwhich represents a hepatic manifestation of a vascular disorder such asobstruction of either the intrahepatic or extrahepatic bile flow or analteration in hepatic circulation resulting, for example, from chronicheart failure, veno-occlusive disease, portal vein thrombosis orBudd-Chiari syndrome.

Additionally, the molecules of the invention can play an important rolein the etiology of certain viral diseases, including but not limited toHepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators ofthe activity of the molecules of the invention could be used to controlviral diseases. The modulators can be used in the treatment and/ordiagnosis of viral infected tissue or virus-associated tissue fibrosis,especially liver and liver fibrosis. Also, such modulators can be usedin the treatment and/or diagnosis of virus-associated carcinoma,especially hepatocellular cancer.

As used herein, a “viral pathogen” or “viral pathogen disorder” includesrespiratory viral pathogens and their associated disorders include, forexample, adenovirus, resulting in upper and lower respiratory tractinfections; conjuctivitis and diarrhea; echovirus, resulting in upperrespiratory tract infections, pharyngitis and rash; rhinovirus,resulting in upper respiratory tract infections; cosackievirus,resulting in Pleurodynia, herpangia, hand-foot-mouth disease;coronavirus, resulting in upper respiratory tract infections; influenzaA and B viruses, resulting in influenza; parainfluenza virus 1-4,resulting in upper and lower respiratory tract infections and croup;respiratory syncytial virus, resulting in bronchiolitis and pneumonia.Digestive viral pathogens and their associated disorders include, forexample, mumps virus, resulting in mumps, pancreatitis, and orchitis;rotavirus, resulting in childhood diarrhea; Norwalk Agent, resulting ingastroenteritis; hepatitis A virus, resulting in acute viral hepatitis;hepatitis B virus, hepatitis D virus and hepatitis C virus, resulting inacute or chronic hepatitis; hepatitis E virus, resulting in entericallytransmitted hepatitis. Systemic viral pathogens associated withdisorders involving skin eruptions include, for example, measles virus,resulting in measles (rubeola); rubella virus, resulting in Germanmeasles (rubella); parvovirus, resulting in erythema infectiosum andaplastic anemia; varicella-zoster virus, resulting in chicken pox andshingles; herpes simplex virus 1-associated, resulting in cold sores;and herpes simplex virus 2, resulting in genital herpes. Systemic viralpathogens associated with hematopoietic disorders include, for example,cytomegalovirus, resulting in cytomegalic inclusion disease;Epstein-Barr virus, resulting in mononucleosis; HTLV-1, resulting inadult T-cell leukemia and tropical spastic paraparesis; HTLV-II; and HIV1 and HIV 2, resulting in AIDS. Arboviral pathogens associated withhemorrhagic fevers include, for example, dengue virus 1-4, resulting indengue and hemorrhagic fever; yellow fever virus, resulting in yellowfever; Colorado tick fever virus, resulting in Colorado tick fever; andregional hemorrhagic fever viruses, resulting in Bolivian, Argentinian,Lassa fever. Viral pathogens associated with warty growths and otherhyperplasias include, for example, papillomavirus, resulting incondyloma and cervical carcinoma; and molluscum virus, resulting inmolluscum contagiosum. Viral pathogens associated with central nervoussystem disorders include, for example, poliovirus, resulting inpoliomyelitis; rabiesvirus, associated with rabies; JC virus, associatedwith progressive multifocal leukoencephalophathy; and arboviralencephalitis viruses, resulting in Eastern, Western, Venezuelan, St.Louis, or California group encephalitis. Viral pathogens associated withcancer include, for example, human papillomaviruses, implicated in thegenesis of several cancers including squamous cell carcinoma of thecervix and anogenital region, oral cancer and laryngeal cancers;Epstein-Barr virus, implicated in pathogenesis of the African form ofBurkitt lymphoma, B-cell lymphomas, Hodgkin disease, and nasopharyngealcarcinomas; hepatitis B virus, implicated in liver cancer; human T-cellleukemia virus type 1 (HTLV-1), associated with T-cellleukemia/lymphoma; and the Kaposi sarcoma herpesvirus (KSHV).

Disorders related to reduced platelet number, thrombocytopenia, includeidiopathic thrombocytopenic purpura, including acute idiopathicthrombocytopenic purpura, drug-induced thrombocytopenia, HIV-associatedthrombocytopenia, and thrombotic microangiopathies: thromboticthrombocytopenic purpura and hemolytic-uremic syndrome.

As used herein, neurological disorders include disorders of the centralnervous system (CNS) and the peripheral nervous system, e.g., cognitiveand neurodegenerative disorders, Examples of neurological disordersinclude, but are not limited to, autonomic function disorders such ashypertension and sleep disorders, and neuropsychiatric disorders, suchas depression, schizophrenia, schizoaffective disorder, Korsakoff'spsychosis, alcoholism, anxiety disorders, or phobic disorders; learningor memory disorders, e.g., amnesia or age-related memory loss, attentiondeficit disorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. Suchneurological disorders include, for example, disorders involvingneurons, and disorders involving glia, such as astrocytes,oligodendrocytes, ependymal cells, and microglia; cerebral edema, raisedintracranial pressure and herniation, and hydrocephalus; malformationsand developmental diseases, such as neural tube defects, forebrainanomalies, posterior fossa anomalies, and syringomyelia and hydromyelia;perinatal brain injury; cerebrovascular diseases, such as those relatedto hypoxia, ischemia, and infarction, including hypotension,hypoperfusion, and low-flow states—global cerebral ischemia and focalcerebral ischemia—infarction from obstruction of local blood supply,intracranial hemorrhage, including intracerebral (intraparenchymal)hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, andvascular malformations, hypertensive cerebrovascular disease, includinglacunar infarcts, slit hemorrhages, and hypertensive encephalopathy;infections, such as acute meningitis, including acute pyogenic(bacterial) meningitis and acute aseptic (viral) meningitis, acute focalsuppurative infections, including brain abscess, subdural empyema, andextradural abscess, chronic bacterial meningoencephalitis, includingtuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis(Lyme disease), viral meningoencephalitis, including arthropod-borne(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplexvirus Type 2, Varicella-zoster virus (Herpes zoster), cytomegalovirus,poliomyelitis, rabies, and human immunodeficiency virus 1, includingHIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer's disease and Pick'sdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson's disease (paralysisagitans) and other Lewy diffuse body diseases, progressive supranuclearpalsy, corticobasal degenration, multiple system atrophy, includingstriatonigral degenration, Shy-Drager syndrome, and olivopontocerebellaratrophy, and Huntington's disease, senile dementia, Gilles de laTourette's syndrome, epilepsy, and Jakob-Creutzfieldt disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease. Further CNS-related disorders include, for example, thoselisted in the American Psychiatric Association's Diagnostic andStatistical manual of Mental Disorders (DSM), the most current versionof which is incorporated herein by reference in its entirety.

As used herein, diseases of the skin, include but are not limited to,disorders of pigmentation and melanocytes, including but not limited to,vitiligo, freckle, melasma, lentigo, nevocellular nevus, dysplasticnevi, and malignant melanoma; benign epithelial tumors, including butnot limited to, seborrheic keratoses, acanthosis nigricans,fibroepithelial polyp, epithelial cyst, keratoacanthoma, and adnexal(appendage) tumors; premalignant and malignant epidermal tumors,including but not limited to, actinic keratosis, squamous cellcarcinoma, basal cell carcinoma, and merkel cell carcinoma; tumors ofthe dermis, including but not limited to, benign fibrous histiocytoma,dermatofibrosarcoma protuberans, xanthomas, and dermal vascular tumors;tumors of cellular immigrants to the skin, including but not limited to,histiocytosis X, mycosis fungoides (cutaneous T-cell lymphoma), andmastocytosis; disorders of epidermal maturation, including but notlimited to, ichthyosis; acute inflammatory dermatoses, including but notlimited to, urticaria, acute eczematous dermatitis, and erythemamultiforme; chronic inflammatory dermatoses, including but not limitedto, psoriasis, lichen planus, and lupus erythematosus; blistering(bullous) diseases, including but not limited to, pemphigus, bullouspemphigoid, dermatitis herpetiformis, and noninflammatory blisteringdiseases: epidermolysis bullosa and porphyria; disorders of epidermalappendages, including but not limited to, acne vulgaris; panniculitis,including but not limited to, erythema nodosum and erythema induratum;and infection and infestation, such as verrucae, molluscum contagiosum,impetigo, superficial fungal infections, and arthropod bites, stings,and infestations.

Additionally, molecules of the invention can play an important role inthe regulation of metabolism or pain disorders. Diseases of metabolicimbalance include, but are not limited to, obesity, anorexia nervosa,cachexia, lipid disorders, and diabetes. Examples of pain disordersinclude, but are not limited to, pain response elicited during variousforms of tissue injury, e.g., inflammation, infection, and ischemia,usually referred to as hyperalgesia (described in, for example, Fields(1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletaldisorders, e.g., joint pain; tooth pain; headaches; pain associated withsurgery; pain related to irritable bowel syndrome; or chest pain.

As used herein, a “hematological disorder” includes a disease, disorder,or condition which affects a hematopoietic cell or tissue. Hematologicaldisorders include diseases, disorders, or conditions associated withaberrant hematological content or function. Hematological disorders canbe characterized by a misregulation (e.g., downregulation orupregulation) of activity. Examples of hematological disorders includedisorders resulting from bone marrow irradiation or chemotherapytreatments for cancer, disorders such as Pernicious Anemia, HemorrhagicAnemia, Hemolytic Anemia, Aplastic Anemia, Sickle Cell Anemia,Sideroblastic Anemia, Anemia associated with chronic infections such asMalaria, Trypanosomiasis, HIV, Hepatitis virus or other viruses,Myelophthisic Anemias caused by marrow deficiencies, renal failureresulting from Anemia, Anemia, Polycethemia, Infectious Mononucleosis(IM), Acute Non-Lymphocytic Leukemia (ANLL), Acute Myeloid Leukemia(AML), Acute Promyelocytic Leukemia (APL), Acute Myelomonocytic Leukemia(AMMoL), Polycethemia Vera, Lymphoma, Acute Lymphocytic Leukemia (ALL),Chronic Lymphocytic Leukemia, Wilm's Tumor, Ewing's Sarcoma,Retinoblastoma, Hemophilia, disorders associated with an increased riskof Thrombosis, Herpes, Thalessemia, antibody-mediated disorders such astransfusion reactions and Erythroblastosis, mechanical trauma to redblood cells such as micro-angiopathic hemolytic anemias, ThromboticThrombocytopenic Purpura and disseminated intravascular coagulation,infections by parasites such as Plasmodium, chemical injuries from,e.g., lead poisoning, and Hypersplenism.

As used herein, the term “hematopoietic cell” includes yolk sac stemcells, primitive erythroid cells, fetal liver cells, fetal spleen cells,fetal bone marrow cells, non-fetal bone marrow cells, megakaryocytes,stem cells, lymphoid stem cells, myeloid stem cells, progenitor cells,progenitor lymphocytes, progenitor T lymphocytes, progenitor Blymphocytes, progenitor erythrocytes, progenitor neutrophils, progenitoreosinophils, progenitor basophils, progenitor monocytes, progenitor mastcells, progenitor platelets, committed lymphocytes, committed Tlymphocytes, committed B lymphocytes, committed erythrocytes, committedneutrophils, committed eosinophils, committed basophils, committedmonocytes, committed mast cells, committed platelets, differentiatedlymphocytes, differentiated T lymphocytes, differentiated B lymphocytes,differentiated erythrocytes, differentiated neutrophils, differentiatedeosinophils, differentiated basophils, differentiated monocytes,differentiated mast cells, differentiated platelets, mature lymphocytes,mature T lymphocytes, mature B lymphocytes, mature erythrocytes, matureneutrophils, mature eosinophils, mature basophils, mature monocytes,mature mast cells, and mature platelets.

As used herein, the term “progenitor cell” includes any somatic cellwhich has the capacity to generate fully differentiated, functionalprogeny by differentiation and proliferation. Progenitor cells includeprogenitors from any tissue or organ system, including, but not limitedto, blood, nerve, muscle, skin, gut, bone, kidney, liver, pancreas,thymus, and the like. Progenitor cells are distinguished from “committedcells” and “differentiated cells,” which are defined as those cellswhich may or may not have the capacity to proliferate, i.e.,self-replicate, but which are unable to undergo further differentiationto a different cell type under normal physiological conditions.Moreover, progenitor cells are further distinguished from abnormal cellssuch as cancer cells, especially leukemia cells, which proliferate(self-replicate) but which generally do not further differentiate,despite appearing to be immature or undifferentiated.

Progenitor cells include all the cells in a lineage of differentiationand proliferation prior to the most differentiated or the fully maturecell. Thus, for example, progenitors include the skin progenitor in themature individual, which is capable of differentiation to only one typeof cell, but which is itself not fully mature or fully differentiated.Production of mature, functional blood cells results from proliferationand differentiation of “unipotential progenitors,” i.e., thoseprogenitors which have the capacity to make only one type of one type ofblood cell. For red blood cell production, a progenitor called a “CFU-E”(colony forming unit-erythroid) has the capacity to generate two to 32progeny cells.

Various other hematopoietic progenitors have been characterized. Forexample, hematopoietic progenitor cells include those cells which arecapable of successive cycles of differentiating and proliferating toyield up to eight different mature hematopoietic cell lineages. At themost primitive or undifferentiated end of the hematopoietic spectrum,hematopoietic progenitor cells include the hematopoietic “stem cells.”These rare cells, which represent 1 in 10,000 to 1 in 100,000 of cellsin the bone marrow, each have the capacity to generate a billion matureblood cells of all lineages and are responsible for sustaining bloodcell production over the life of an animal. They reside in the marrowprimarily in a quiescent state and may form identical daughter cellsthrough a process called self-renewal. Accordingly, such an uncommittedprogenitor can be described as being “totipotent,” i.e., both necessaryand sufficient for generating all types of mature blood cells.Progenitor cells which retain a capacity to generate all blood celllineages but which can not self-renew are termed “pluripotent.” Cellswhich can produce some but not all blood lineages and can not self-reneware termed “multipotent.”

As used herein, “hematopoietic cell activity” includes an activityexerted by a hematopoietic cell, or an activity that takes place in ahematopoietic cell. For example, such activities include cellularprocesses that contribute to the physiological role of hematopoieticcells, such as hematopoiesis, but are not limited to, cellproliferation, differentiation, growth, migration and programmed celldeath.

As used herein, the term “erythroid associated disorders” includedisorders involving aberrant (increased or deficient) erythroblastproliferation, e.g., an erythroleukemia, and aberrant (increased ordeficient) erythroblast differentiation, e.g., an anemia.Erythrocyte-associated disorders include anemias such as, for example,drug-(chemotherapy-) induced anemias, hemolytic anemias due tohereditary cell membrane abnormalities, such as hereditaryspherocytosis, hereditary elliptocytosis, and hereditarypyropoikilocytosis; hemolytic anemias due to acquired cell membranedefects, such as paroxysmal nocturnal hemoglobinuria and spur cellanemia; hemolytic anemias caused by antibody reactions, for example tothe RBC antigens, or antigens of the ABO system, Lewis system, Iisystem, Rh system, Kidd system, Duffy system, and Kell system;methemoglobinemia; a failure of erythropoiesis, for example, as a resultof aplastic anemia, pure red cell aplasia, myelodysplastic syndromes,sideroblastic anemias, and congenital dyserythropoietic anemia;secondary anemia in non-hematolic disorders, for example, as a result ofchemotherapy, alcoholism, or liver disease; anemia of chronic disease,such as chronic renal failure; and endocrine deficiency diseases.

Anemias are an important class of disorders which affect erythroid cellgrowth or differentiation. Anemias include a variety of disorders inwhich the content of erythrocytes or hemoglobin in the blood isinsufficient to fully provide transportation of oxygen for all of thebody's needs. Examples of anemias include hemolytic anemias attributableto hereditary cell membrane abnormalities (e.g., hereditaryspherocytosis, hereditary elliptocytosis, and hereditarypyropoikilocytosis), hemolytic anemias attributable to acquired cellmembrane defects (e.g., paroxysmal nocturnal hemoglobinuria and spurcell anemia), hemolytic anemias attributable to antibody reactions(e.g., reactions to RBC antigens or antigens of the ABO system, Lewissystem, Ii system, Rh system, Kidd system, Duffy system, and Kellsystem), methemoglobinemia, anemias attributable to a failure oferythropoiesis (e.g., as a result of aplastic anemia, pure red cellaplasia, myelodysplastic syndromes, sideroblastic anemias, or congenitaldyserythropoietic anemia), secondary anemia in non-hematologicaldisorders (e.g., anemia attributable to chemotherapy, alcoholism, orliver disease), anemia associated with a chronic disease (e.g., anemiaassociated with chronic renal failure), anemias attributable tobleeding, and anemia associated with endocrine deficiency diseases. Someof these disorders are known to affect growth or differentiation oferythroid cells.

As used herein, the term “modulate” includes alteration of, e.g., byincreasing or decreasing the particular parameter being described.

As used herein, the term “apoptosis” includes programmed cell deathwhich can be characterized using techniques which are known in the art.Apoptotic cell death can be characterized, e.g., by cell shrinkage,membrane blebbing and chromatin condensation culminating in cellfragmentation. Cells undergoing apoptosis also display a characteristicpattern of internucleosomal DNA cleavage.

As used herein, the term “endocrine disorder” includes, but is notlimited to, disorders of the pancreas, e.g., diabetes mellitus; isletcell tumors, including but not limited to, insulinomas, gastrinomas, andother rare islet cell tumors, the pituitary, or the hypothalamus.

Various aspects of the invention are described in further detail below.

Isolated Nucleic Acid Molecules

In one aspect, the invention provides, an isolated or purified, nucleicacid molecule that encodes a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 polypeptide described herein, e.g.,a full length 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein or a fragment thereof, e.g., abiologically active portion of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein. Also included is a nucleicacid fragment suitable for use as a hybridization probe, which can beused, e.g., to identify a nucleic acid molecule encoding a polypeptideof the invention, 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 mRNA, and fragments suitable for use asprimers, e.g., PCR primers for the amplification or mutation of nucleicacid molecules.

In one embodiment, an isolated nucleic acid molecule of the inventionincludes the nucleotide sequence shown in SEQ ID NO: 1, 3, 4, 6, 11, 13,20, 22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75, 76, 78, 86 or88, or a portion of any of this nucleotide sequence. In one embodiment,the nucleic acid molecule includes sequences encoding the human 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577protein (i.e., “the coding region” of SEQ ID NO: 1, 4, 11, 20, 25, 29,35, 38, 41, 73, 76 or 86, as shown in SEQ ID NO: 3, 6, 13, 22, 27, 31,37, 40, 43, 75, 78 or 88, respectively), as well as 5′ untranslatedsequences and 3′ untranslated sequences. Alternatively, the nucleic acidmolecule can include only the coding region of SEQ ID NO: 1, 4, 11, 20,25, 29, 35, 38, 41, 73, 76 or 86 (e.g., SEQ ID NO: 3, 6, 13, 22, 27, 31,37, 40, 43, 75, 78 or 88) and, e.g., no flanking sequences whichnormally accompany the subject sequence. In another embodiment, thenucleic acid molecule encodes a sequence corresponding to a fragment ofthe protein corresponding to conserved domains identified within SEQ IDNO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87.

In another embodiment, an isolated nucleic acid molecule of theinvention includes a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO: 1, 3, 4, 6, 11, 13, 20, 22, 25,27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75, 76, 78, 86 or 88, or aportion of any of these nucleotide sequences. In other embodiments, thenucleic acid molecule of the invention is sufficiently complementary tothe nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 11, 13, 20, 22,25, 27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75, 76, 78, 86 or 88 suchthat it can hybridize to the nucleotide sequence shown in SEQ ID NO: 1,3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75,76, 78, 86 or 88, thereby forming a stable duplex.

In one embodiment, an isolated nucleic acid molecule of the presentinvention includes a nucleotide sequence which is at least about: 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more homologous to the entire length of the nucleotide sequenceshown in SEQ ID NO: 1, 3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37,38, 40, 41, 43, 73, 75, 76, 78, 86 or 88, or a portion, preferably ofthe same length, of any of these nucleotide sequences.

27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 Nucleic Acid Fragments

A nucleic acid molecule of the invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:1, 3, 4, 6, 11, 13, 20, 22, 25,27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75, 76, 78, 86 or 88. Forexample, such a nucleic acid molecule can include a fragment which canbe used as a probe or primer or a fragment encoding a portion of a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein, e.g., an immunogenic or biologically active portion of a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein. A fragment can comprise those nucleotides of SEQ ID NO:1,3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75,76, 78, 86 or 88, which encode a domain of human 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577. The nucleotidesequence determined from the cloning of the 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene allows for thegeneration of probes and primers designed for use in identifying and/orcloning other 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 family members, or fragments thereof, as well as27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 homologs, or fragments thereof, from other species.

In another embodiment, a nucleic acid includes a nucleotide sequencethat includes part, or all, of the coding region and extends into either(or both) the 5′ or 3′ noncoding region. Other embodiments include afragment which includes a nucleotide sequence encoding an amino acidfragment described herein. Nucleic acid fragments can encode a specificdomain or site described herein or fragments thereof, particularlyfragments thereof which are at least 100 amino acids in length.Fragments also include nucleic acid sequences corresponding to specificamino acid sequences described above or fragments thereof. Nucleic acidfragments should not to be construed as encompassing those fragmentsthat may have been disclosed prior to the invention.

A nucleic acid fragment can include a sequence corresponding to adomain, region, or functional site described herein. A nucleic acidfragment can also include one or more domain, region, or functional sitedescribed herein. Thus, for example, a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 nucleic acid fragmentcan include a sequence corresponding to a domain of 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577, as describedherein.

27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 probes and primers are provided. Typically a probe/primer is anisolated or purified oligonucleotide. The oligonucleotide typicallyincludes a region of nucleotide sequence that hybridizes under stringentconditions to at least about 7, 12 or 15, preferably about 20 or 25,more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutivenucleotides of a sense or antisense sequence of SEQ ID NO:1, 3, 4, 6,11, 13, 20, 22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75, 76, 78,86 or 88, or of a naturally occurring allelic variant or mutant of SEQID NO:1, 3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37, 38, 40, 41,43, 73, 75, 76, 78, 86 or 88.

In a preferred embodiment the nucleic acid is a probe which is at least5 or 10, and less than 200, more preferably less than 100, or less than50, base pairs in length. It should be identical, or differ by 1, orless than in 5 or 10 bases, from a sequence disclosed herein. Ifalignment is needed for this comparison the sequences should be alignedfor maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.

A probe or primer can be derived from the sense or anti-sense strand ofa nucleic acid which encodes a domain identified in the 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577sequences.

In another embodiment a set of primers is provided, e.g., primerssuitable for use in a PCR, which can be used to amplify a selectedregion of a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 sequence, e.g., a domain, region, site or othersequence described herein. The primers should be at least 5, 10, or 50base pairs in length and less than 100, or less than 200, base pairs inlength. The primers should be identical, or differ by one base from asequence disclosed herein or from a naturally occurring variant.

A nucleic acid fragment can encode an epitope bearing region of apolypeptide described herein.

A nucleic acid fragment encoding a “biologically active portion of a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 polypeptide” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO:1, 3, 4, 6, 11, 13, 20, 22, 25, 27, 29,31, 35, 37, 38, 40, 41, 43, 73, 75, 76, 78, 86 or 88, which encodes apolypeptide having a 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 biological activity (e.g., the biologicalactivities of the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 proteins are described herein), expressingthe encoded portion of the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein (e.g., by recombinantexpression in vitro) and assessing the activity of the encoded portionof the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 protein. A nucleic acid fragment encoding a biologicallyactive portion of a 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 polypeptide, can comprise a nucleotidesequence which is greater than 300 or more nucleotides in length.

In preferred embodiments, a nucleic acid includes a nucleotide sequencewhich is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400,2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600,3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800,4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000,6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200,or more nucleotides in length and hybridizes under stringenthybridization conditions to a nucleic acid molecule of SEQ ID NO:1, 3,4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75,76, 78, 86 or 88.

27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 Nucleic Acid Variants

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 11, 13, 20,22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43, 73, 75, 76, 78, 86 or 88.Such differences can be due to degeneracy of the genetic code (andresult in a nucleic acid which encodes the same 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteins asthose encoded by the nucleotide sequence disclosed herein. In anotherembodiment, an isolated nucleic acid molecule of the invention has anucleotide sequence encoding a protein having an amino acid sequencewhich differs, by at least 1, but less than 5, 10, 20, 50, or 100 aminoacid residues that shown in SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42,74, 77 or 87. If alignment is needed for this comparison the sequencesshould be aligned for maximum homology. “Looped” out sequences fromdeletions or insertions, or mismatches, are considered differences.

Nucleic acids of the inventor can be chosen for having codons, which arepreferred, or non-preferred, for a particular expression system. E.g.,the nucleic acid can be one in which at least one codon, at preferablyat least 10%, or 20% of the codons has been altered such that thesequence is optimized for expression in E. coli, yeast, human, insect,or CHO cells.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologs (different locus), and orthologs(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

In a preferred embodiment, the nucleic acid differs from that of SEQ IDNO:1, 3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43,73, 75, 76, 78, 86 or 88, e.g., as follows: by at least one but lessthan 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%,10% or 20% of the nucleotides in the subject nucleic acid. If necessaryfor this analysis the sequences should be aligned for maximum homology.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences.

Orthologs, homologs, and allelic variants can be identified usingmethods known in the art. These variants comprise a nucleotide sequenceencoding a polypeptide that is 50%, at least about 55%, typically atleast about 70-75%, more typically at least about 80-85%, and mosttypically at least about 90-95% or more identical to the nucleotidesequence shown in SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or87 or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under stringentconditions, to the nucleotide sequence shown in SEQ ID NO:2, 5, 12, 21,26, 30, 36, 39, 42, 74, 77 or 87 or a fragment of the sequence. Nucleicacid molecules corresponding to orthologs, homologs, and allelicvariants of the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 cDNAs of the invention can further be isolated bymapping to the same chromosome or locus as the 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene. Preferredvariants include those that are correlated with activities specific tothe molecules of the invention, i.e. phospholipase activity, serinecarboxypeptidase activity, trypsin-like serine protease activity,aldehyde dehydrogenase activity, ubiquitin-protein ligase activity,protein kinase activity, hydrolase activity or matrix metalloproteinaseactivity.

Allelic variants of 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577, e.g., human 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577, include bothfunctional and non-functional proteins. Functional allelic variants arenaturally occurring amino acid sequence variants of the 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteinwithin a population that maintain the ability to bind a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 ligandor substrate and/or modulate phospholipase activity, serinecarboxypeptidase activity, trypsin-like serine protease activity,aldehyde dehydrogenase activity, ubiquitin-protein ligase activity,protein kinase activity, hydrolase activity or matrix metalloproteinaseactivity. Functional allelic variants will typically contain onlyconservative substitution of one or more amino acids of SEQ ID NO:2, 5,12, 21, 26, 30, 36, 39, 42, 74, 77 or 87, or substitution, deletion orinsertion of non-critical residues in non-critical regions of theprotein. Non-functional allelic variants are naturally-occurring aminoacid sequence variants of the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577, e.g., human 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577, protein withina population that do not have the ability to bind a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 ligand orsubstrate and/or phospholipase activity, serine carboxypeptidaseactivity, trypsin-like serine protease activity, aldehyde dehydrogenaseactivity, ubiquitin-protein ligase activity, protein kinase activity,hydrolase activity or matrix metalloproteinase activity. Non-functionalallelic variants will typically contain a non-conservative substitution,a deletion, or insertion, or premature truncation of the amino acidsequence of SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87, ora substitution, insertion, or deletion in critical residues or criticalregions of the protein.

Moreover, nucleic acid molecules encoding other 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 family membersand, thus, which have a nucleotide sequence which differs from the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 sequences of SEQ ID NO:1, 3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31,35, 37, 38, 40, 41, 43, 73, 75, 76, 78, 86 or 88 are intended to bewithin the scope of the invention.

Antisense Nucleic Acid Molecules, Ribozymes and Modified 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 NucleicAcid Molecules

In another aspect, the invention features, an isolated nucleic acidmolecule which is antisense to 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577. An “antisense” nucleic acid caninclude a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. The antisense nucleic acid can be complementary to an entire27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 coding strand, or to only a portion thereof (e.g., the codingregion of human 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 corresponding to SEQ ID NO:3, 6, 13, 22, 27, 31,37, 40, 43, 75, 78, 88, respectively). In another embodiment, theantisense nucleic acid molecule is antisense to a “noncoding region” ofthe coding strand of a nucleotide sequence encoding 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 (e.g., the 5′and 3′ untranslated regions).

An antisense nucleic acid can be designed such that it is complementaryto the entire coding region of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 mRNA, e.g., between the −10 and +10regions of the target gene nucleotide sequence of interest. An antisenseoligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. The antisense nucleic acid also canbe produced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject (e.g., by direct injection at a tissue site),or generated in situ such that they hybridize with or bind to cellularmRNA and/or genomic DNA encoding a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein to thereby inhibitexpression of the protein, e.g., by inhibiting transcription and/ortranslation. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For systemic administration, antisense molecules can be modified suchthat they specifically or selectively bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. A ribozyme having specificity for a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577-encodingnucleic acid can include one or more sequences complementary to thenucleotide sequence of a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 cDNA disclosed herein (i.e., SEQ IDNO: 1, 3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35, 37, 38, 40, 41, 43,73, 75, 76, 78, 86 or 88), and a sequence having known catalyticsequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 orHaselhoff and Gerlach (1988) Nature 334:585-591). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577-encoding mRNA. See,e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.5,116,742. Alternatively, 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 mRNA can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418.

27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 gene expression can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 (e.g., the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 promoter and/or enhancers) to form triple helical structures thatprevent transcription of the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 gene in target cells. See generally,Helene (1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N.Y.Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:807-15. Thepotential sequences that can be targeted for triple helix formation canbe increased by creating a so-called “switchback” nucleic acid molecule.Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′manner, such that they base pair with first one strand of a duplex andthen the other, eliminating the necessity for a sizeable stretch ofeither purines or pyrimidines to be present on one strand of a duplex.

The invention also provides detectably labeled oligonucleotide primerand probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or calorimetric.

A 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 nucleic acid molecule can be modified at the base moiety, sugarmoiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For example, thedeoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup et al. (1996)Bioorganic & Medicinal Chemistry 4: 5-23).

As used herein, the terms “peptide nucleic acid” or “PNA” refers to anucleic acid mimic, e.g., a DNA mimic, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of a PNA canallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.93: 14670-675.

PNAs of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 nucleic acid molecules can also be used inthe analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., S1 nucleases (Hyrup etal. (1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup et al. (1996) supra; Perry-O'Keefe supra).

In other embodiments, the oligonucleotide can include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide can be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

The invention also includes molecular beacon oligonucleotide primer andprobe molecules having at least one region which is complementary to a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 nucleic acid of the invention, two complementary regions onehaving a fluorophore and one a quencher such that the molecular beaconis useful for quantitating the presence of the 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 nucleic acid ofthe invention in a sample. Molecular beacon nucleic acids are described,for example, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko etal., U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

Isolated 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 Polypeptides

In another aspect, the invention features, an isolated 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein,or fragment, e.g., a biologically active portion, for use as immunogensor antigens to raise or test (or more generally to bind) anti-27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577antibodies. 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein can be isolated from cells or tissuesources using standard protein purification techniques. 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteinor fragments thereof can be produced by recombinant DNA techniques orsynthesized chemically.

Polypeptides of the invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events,alternative RNA splicing events, and alternative translational andpost-translational events. The polypeptide can be expressed in systems,e.g., cultured cells, which result in substantially the samepost-translational modifications present when the polypeptide isexpressed in a native cell, or in systems which result in the alterationor omission of post-translational modifications, e.g., glycosylation orcleavage, present in a native cell.

In a preferred embodiment, a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 polypeptide has one or more of thefollowing characteristics:

it has the ability to catalyze the hydrolysis of an acyl or phosphoacylbond of a phospholipids; to modulate removal of COOH-terminal residues;(ii) it has the ability to modulate the transfer of an acyl group from adonor to an acceptor molecule; (iii) it has the ability to phosphorylatecarbohydrates; (iv) it has the ability to oxidate an aldehyde; (v) ithas the ability to modulate ubiquitination of a substrate; (vi) it hasthe ability to reversibly phosphorylate proteins in order to regulateprotein activity in eukaryotic cells; (vii) it has the ability tointeract with cytotoxins and metabolites; (viii) it has the ability tocatalyze the metabolism of a cytotoxin or metabolite; (ix) it has theability to hydrolyze a thioester containing compound; (x) it has theability to catalyze the formation of a thioester conjugation on asubstrate; (xi) it has the ability to cleave or modulate the degredationof proteins or peptides of the extracellular matrix; (xii) it has theability to catalyze or modulate catalysis of cleavage of covalent bondswithin or between amino acid residues; (xiii) it has a molecular weight,e.g., a deduced molecular weight, preferably ignoring any contributionof post translational modifications, amino acid composition or otherphysical characteristic of a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 polypeptide, e.g., a polypeptide ofSEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87; (xiv) it hasan overall sequence similarity of at least 60%, preferably at least 70%,more preferably at least 80, 90, or 95%, with a polypeptide of SEQ IDNO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87; (xv) it is expressedin a multitude of human tissues and cell lines (refer to section foreach molecule of the invention); and it has specific domains which arepreferably about 70%, 80%, 90% or 95% identical to the identified aminoacid residues of SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or87 (refer to section for each molecule of the invention for domain namesand locations within amino acid sequence).

In a preferred embodiment the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein, or fragment thereof,differs from the corresponding sequence in SEQ ID NO:2, 5, 12, 21, 26,30, 36, 39, 42, 74, 77 or 87. In one embodiment it differs by at leastone but by less than 15, 10 or 5 amino acid residues. In another itdiffers from the corresponding sequence in SEQ ID NO:2, 5, 12, 21, 26,30, 36, 39, 42, 74, 77 or 87 by at least one residue but less than 20%,15%, 10% or 5% of the residues in it differ from the correspondingsequence in SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87.(If this comparison requires alignment the sequences should be alignedfor maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.) The differencesare, preferably, differences or changes at a non-essential residue or aconservative substitution. In a preferred embodiment the differences arenot in the identified or conserved domain(s) within SEQ ID NO:2, 5, 12,21, 26, 30, 36, 39, 42, 74, 77 or 87. In another embodiment one or moredifferences are in the identified or conserved domain(s) within SEQ IDNO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87.

Other embodiments include a protein that contains one or more changes inamino acid sequence, e.g., a change in an amino acid residue which isnot essential for activity. Such 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 proteins differ in amino acidsequence from SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87,yet retain biological activity.

In one embodiment, the protein includes an amino acid sequence at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous toSEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87.

A 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 protein or fragment is provided which varies from the sequenceof SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87 in regionsdefined by amino acids that are not within identified or conserveddomains or regions by at least one but by less than 15, 10 or 5 aminoacid residues in the protein or fragment but which does not differ fromSEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87 in regionsdefined by amino acids that are within identified or conserved domainsor regions. (If this comparison requires alignment the sequences shouldbe aligned for maximum homology. “Looped” out sequences from deletionsor insertions, or mismatches, are considered differences.) In someembodiments the difference is at a non-essential residue or is aconservative substitution, while in others the difference is at anessential residue or is a non-conservative substitution.

In one embodiment, a biologically active portion of a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteinincludes an identified domain (refer to section for each molecule of theinvention). Moreover, other biologically active portions, in which otherregions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofa native 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 protein.

In a preferred embodiment, the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein has an amino acid sequenceshown in SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87. Inother embodiments, the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 protein is sufficiently or substantiallyidentical to SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87.In yet another embodiment, the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein is sufficiently orsubstantially identical to SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42,74, 77 or 87 and retains the functional activity of the protein of SEQID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87, as described indetail in the subsections above.

27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 Chimeric or Fusion Proteins

In another aspect, the invention provides 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 chimeric or fusionproteins. As used herein, a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 “chimeric protein” or “fusionprotein” includes a 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 polypeptide linked to a non-27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577polypeptide. A “non-27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 polypeptide” refers to a polypeptide havingan amino acid sequence corresponding to a protein which is notsubstantially homologous to the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein, e.g., a proteinwhich is different from the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein and which is derived fromthe same or a different organism. The 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 polypeptide of the fusionprotein can correspond to all or a portion e.g., a fragment describedherein of a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 amino acid sequence. In a preferred embodiment, a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 fusion protein includes at least one (or two) biologically activeportion of a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein. The non-27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptide can befused to the N-terminus or C-terminus of the 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptide.

The fusion protein can include a moiety which has a high affinity for aligand. For example, the fusion protein can be a GST-27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 fusionprotein in which the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 sequences are fused to the C-terminus ofthe GST sequences. Such fusion proteins can facilitate the purificationof recombinant 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577. Alternatively, the fusion protein can be a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577protein containing a heterologous signal sequence at its N-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 can be increased through use of a heterologoussignal sequence.

Fusion proteins can include all or a part of a serum protein, e.g., aportion of an immunoglobulin (e.g., IgG, IgA, or IgE), e.g., an Fcregion and/or the hinge C1 and C2 sequences of an immunoglobulin orhuman serum albumin.

The 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. The27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 fusion proteins can be used to affect the bioavailability of a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 substrate. 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 fusion proteins can be useful therapeutically forthe treatment of disorders caused by, for example, (i) aberrantmodification or mutation of a gene encoding a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein; (ii)mis-regulation of the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 gene; and (iii) aberrant post-translationalmodification of a 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 protein.

Moreover, the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577-fusion proteins of the invention can be used asimmunogens to produce anti-27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 antibodies in a subject, to purify27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 ligands and in screening assays to identify molecules whichinhibit the interaction of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 with a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 substrate.

Expression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). A 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein.

Variants of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 Proteins

In another aspect, the invention also features a variant of a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577polypeptide, e.g., which functions as an agonist (mimetics) or as anantagonist. Variants of the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 proteins can be generated bymutagenesis, e.g., discrete point mutation, the insertion or deletion ofsequences or the truncation of a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein. An agonist of the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 proteins can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein.An antagonist of a 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 protein can inhibit one or more of theactivities of the naturally occurring form of the 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein by, forexample, competitively modulating a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577-mediated activity of a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577protein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. Preferably, treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein.

Variants of a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein for agonist or antagonist activity.

Libraries of fragments e.g., N terminal, C terminal, or internalfragments, of a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein coding sequence can be used to generate avariegated population of fragments for screening and subsequentselection of variants of a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein.

Variants in which a cysteine residues is added or deleted or in which aresidue which is glycosylated is added or deleted are particularlypreferred.

Methods for screening gene products of combinatorial libraries made bypoint mutations or truncation, and for screening cDNA libraries for geneproducts having a selected property are known in the art. Recursiveensemble mutagenesis (REM), a new technique which enhances the frequencyof functional mutants in the libraries, can be used in combination withthe screening assays to identify 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 variants (Arkin and Yourvan(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993)Protein Engineering 6:327-331).

Cell based assays can be exploited to analyze a variegated 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 library.For example, a library of expression vectors can be transfected into acell line, e.g., a cell line, which ordinarily responds to 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 in asubstrate-dependent manner. The transfected cells are then contactedwith 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 and the effect of the expression of the mutant onsignaling by the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 substrate can be detected, e.g., by measuringeither phospholipase activity, serine carboxypeptidase activity,trypsin-like serine protease activity, aldehyde dehydrogenase activity,ubiquitin-protein ligase activity, protein kinase activity, hydrolaseactivity, matrix metalloproteinase activity, or other activity. PlasmidDNA can then be recovered from the cells which score for inhibition, oralternatively, potentiation of signaling by the 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 substrate, andthe individual clones further characterized.

In another aspect, the invention features a method of making a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577polypeptide, e.g., a peptide having a non-wild type activity, e.g., anantagonist, agonist, or super agonist of a naturally occurring 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577polypeptide, e.g., a naturally occurring 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptide. Themethod includes altering the sequence of a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptide, e.g.,altering the sequence, e.g., by substitution or deletion of one or moreresidues of a non-conserved region, a domain or residue disclosedherein, and testing the altered polypeptide for the desired activity.

In another aspect, the invention features a method of making a fragmentor analog of a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 polypeptide a biological activity of a naturallyoccurring 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 polypeptide. The method includes altering the sequence,e.g., by substitution or deletion of one or more residues, of a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577polypeptide, e.g., altering the sequence of a non-conserved region, or adomain or residue described herein, and testing the altered polypeptidefor the desired activity.

Anti-27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 Antibodies

In another aspect, the invention provides an anti-27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 antibody. Theterm “antibody” as used herein refers to an immunoglobulin molecule orimmunologically active portion thereof, i.e., an antigen-bindingportion. Examples of immunologically active portions of immunoglobulinmolecules include scFV and dcFV fragments, Fab and F(ab′)₂ fragmentswhich can be generated by treating the antibody with an enzyme such aspapain or pepsin, respectively.

The antibody can be a polyclonal, monoclonal, recombinant, e.g., achimeric or humanized, fully human, non-human, e.g., murine, or singlechain antibody. In a preferred embodiment it has effector function andcan fix complement. The antibody can be coupled to a toxin or imagingagent.

A full-length 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein or, antigenic peptide fragment of 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577can be used as an immunogen or can be used to identify anti-27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577antibodies made with other immunogens, e.g., cells, membranepreparations, and the like. The antigenic peptide of 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 shouldinclude at least 8 amino acid residues of the amino acid sequence shownin SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87 andencompasses an epitope of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577. Preferably, the antigenic peptideincludes at least 10 amino acid residues, more preferably at least 15amino acid residues, even more preferably at least 20 amino acidresidues, and most preferably at least 30 amino acid residues.

Fragments of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 which include hydrophilic regions of SEQ ID NO:2,5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87 can be used to make, e.g.,used as immunogens or used to characterize the specificity of anantibody, antibodies against hydrophilic regions of the 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein.Similarly, fragments of 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 which include hydrophobic regions of SEQ IDNO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87 can be used to make anantibody against a hydrophobic region of the 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein; fragments of27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 which include residues within extra cellular domain(s) of SEQ IDNO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74, 77 or 87 can be used to make anantibody against an extracellular or non-cytoplasmic region of the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein; fragments of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 which include residues withinintracellular regions of SEQ ID NO:2, 5, 12, 21, 26, 30, 36, 39, 42, 74,77 or 87 can be used to make an antibody against an intracellular regionof the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 protein; a fragment of 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 which include residues withinidentified or conserved domains of SEQ ID NO:2, 5, 12, 21, 26, 30, 36,39, 42, 74, 77 or 87 can be used to make an antibody against theidentified or conserved domain of the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein.

Antibodies reactive with, or specific or selective for, any of theseregions, or other regions or domains described herein are provided.

Preferred epitopes encompassed by the antigenic peptide are regions of27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 located on the surface of the protein, e.g., hydrophilic regions,as well as regions with high antigenicity. For example, an Emini surfaceprobability analysis of the human 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein sequence can be usedto indicate the regions that have a particularly high probability ofbeing localized to the surface of the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein and are thus likelyto constitute surface residues useful for targeting antibody production.

In a preferred embodiment the antibody can bind to the extracellularportion of the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein, e.g., it can bind to a whole cell whichexpresses the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein. In another embodiment, the antibody bindsan intracellular portion of the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein.

In a preferred embodiment the antibody binds an epitope on any domain orregion on 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 proteins described herein.

Additionally, chimeric, humanized, and completely human antibodies arealso within the scope of the invention. Chimeric, humanized, but mostpreferably, completely human antibodies are desirable for applicationswhich include repeated administration, e.g., therapeutic treatment ofhuman patients, and some diagnostic applications.

Chimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, can be made using standard recombinant DNAtechniques. Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in Robinson et al. International Application No.PCT/US86/02269; Akira, et al. European Patent Application 184,187;Taniguchi, European Patent Application 171,496; Morrison et al. EuropeanPatent Application 173,494; Neuberger et al. PCT InternationalPublication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567;Cabilly et al. European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559).

A humanized or complementarity determining region (CDR)-grafted antibodywill have at least one or two, but generally all three recipient CDR's(of heavy and or light immuoglobulin chains) replaced with a donor CDR.The antibody may be replaced with at least a portion of a non-human CDRor only some of the CDR's may be replaced with non-human CDR's. It isonly necessary to replace the number of CDR's required for binding ofthe humanized antibody to a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 or a fragment thereof. Preferably,the donor will be a rodent antibody, e.g., a rat or mouse antibody, andthe recipient will be a human framework or a human consensus framework.Typically, the immunoglobulin providing the CDR's is called the “donor”and the immunoglobulin providing the framework is called the “acceptor.”In one embodiment, the donor immunoglobulin is a non-human (e.g.,rodent). The acceptor framework is a naturally-occurring (e.g., a human)framework or a consensus framework, or a sequence about 85% or higher,preferably 90%, 95%, 99% or higher identical thereto.

As used herein, the term “consensus sequence” refers to the sequenceformed from the most frequently occurring amino acids (or nucleotides)in a family of related sequences (See e.g., Winnaker, (1987) From Genesto Clones (Verlagsgesellschaft, Weinheim, Germany). In a family ofproteins, each position in the consensus sequence is occupied by theamino acid occurring most frequently at that position in the family. Iftwo amino acids occur equally frequently, either can be included in theconsensus sequence. A “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence.

An antibody can be humanized by methods known in the art. Humanizedantibodies can be generated by replacing sequences of the Fv variableregion which are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are provided by Morrison (1985) Science229:1202-1207, by Oi et al. (1986) BioTechniques 4:214, and by Queen etal. U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, the contents ofall of which are hereby incorporated by reference. Those methods includeisolating, manipulating, and expressing the nucleic acid sequences thatencode all or part of immunoglobulin Fv variable regions from at leastone of a heavy or light chain. Sources of such nucleic acid are wellknown to those skilled in the art and, for example, may be obtained froma hybridoma producing an antibody against a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptide orfragment thereof. The recombinant DNA encoding the humanized antibody,or fragment thereof, can then be cloned into an appropriate expressionvector.

Humanized or CDR-grafted antibodies can be produced by CDR-grafting orCDR substitution, wherein one, two, or all CDR's of an immunoglobulinchain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534;Beidler et al. (1988) J. Immunol. 141:4053-4060; Winter U.S. Pat. No.5,225,539, the contents of all of which are hereby expresslyincorporated by reference. Winter describes a CDR-grafting method whichmay be used to prepare the humanized antibodies of the present invention(UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S.Pat. No. 5,225,539), the contents of which is expressly incorporated byreference.

Also within the scope of the invention are humanized antibodies in whichspecific amino acids have been substituted, deleted or added. Preferredhumanized antibodies have amino acid substitutions in the frameworkregion, such as to improve binding to the antigen. For example, ahumanized antibody will have framework residues identical to the donorframework residue or to another amino acid other than the recipientframework residue. To generate such antibodies, a selected, small numberof acceptor framework residues of the humanized immunoglobulin chain canbe replaced by the corresponding donor amino acids. Preferred locationsof the substitutions include amino acid residues adjacent to the CDR, orwhich are capable of interacting with a CDR (see e.g., U.S. Pat. No.5,585,089). Criteria for selecting amino acids from the donor aredescribed in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat.No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, thecontents of which are hereby incorporated by reference. Other techniquesfor humanizing antibodies are described in Padlan et al. EP 519596 A1,published on Dec. 23, 1992.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See, for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Fremont, Calif.) and Medarex, Inc. (Princeton, N.J.), canbe engaged to provide human antibodies directed against a selectedantigen using technology similar to that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

The anti-27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 antibody can be a single chain antibody. A single-chainantibody (scFV) can be engineered as described in, for example, Colcheret al. (1999) Ann. N Y Acad. Sci. 880:263-80; and Reiter (1996) Clin.Cancer Res. 2:245-52. The single chain antibody can be dimerized ormultimerized to generate multivalent antibodies having specificities fordifferent epitopes of the same target 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein.

In a preferred embodiment, the antibody has reduced or no ability tobind an Fc receptor. For example, it is an isotype or subtype, fragmentor other mutant, which does not support binding to an Fc receptor, e.g.,it has a mutagenized or deleted Fc receptor binding region.

An antibody (or fragment thereof) may be conjugated to a therapeuticmoiety such as a cytotoxin, a therapeutic agent or a radioactive ion. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No.5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545)and analogs or homologs thereof. Therapeutic agents include, but are notlimited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine, vinblastine, taxol andmaytansinoids).

Radioactive ions include, but are not limited to iodine, yttrium andpraseodymium.

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, thetherapeutic moiety may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; aprotein such as tumor necrosis factor, α-interferon, β-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

An anti-27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 antibody (e.g., monoclonal antibody) can be used toisolate 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 by standard techniques, such as affinity chromatographyor immunoprecipitation. Moreover, an anti-27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 antibody can be usedto detect 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 protein (e.g., in a cellular lysate or cell supernatant)in order to evaluate the abundance and pattern of expression of theprotein. Anti-27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance (i.e., antibody labelling). Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In preferred embodiments, an antibody can be made by immunizing with apurified 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 antigen, or a fragment thereof, e.g., a fragmentdescribed herein, a membrane associated antigen, tissues, e.g., crudetissue preparations, whole cells, preferably living cells, lysed cells,or cell fractions, e.g., membrane fractions.

Antibodies which bind only a native 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein, only denatured orotherwise non-native 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 protein, or which bind both, are within theinvention. Antibodies with linear or conformational epitopes are withinthe invention. Conformational epitopes sometimes can be identified byidentifying antibodies which bind to native but not denatured 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577protein.

Recombinant Expression Vectors, Host Cells and Genetically EngineeredCells

In another aspect, the invention includes, vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidedescribed herein. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked and can include a plasmid, cosmid or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors include, e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses.

A vector can include a 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 nucleic acid in a form suitable forexpression of the nucleic acid in a host cell.

Preferably the recombinant expression vector includes one or moreregulatory sequences operatively linked to the nucleic acid sequence tobe expressed. The term “regulatory sequence” includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Regulatory sequences include those which direct constitutiveexpression of a nucleotide sequence, as well as tissue-specificregulatory and/or inducible sequences. The design of the expressionvector can depend on such factors as the choice of the host cell to betransformed, the level of expression of protein desired, and the like.The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or polypeptides, including fusionproteins or polypeptides, encoded by nucleic acids as described herein(e.g., 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 proteins, mutant forms of 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteins, fusionproteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 proteins in prokaryotic or eukaryotic cells. Forexample, polypeptides of the invention can be expressed in E. coli,insect cells (e.g., using baculovirus expression vectors), yeast cellsor mammalian cells. Suitable host cells are discussed further inGoeddel, (1990) Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein.

Purified fusion proteins can be used in 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 activity assays,(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific or selective for 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteins. In apreferred embodiment, a fusion protein expressed in a retroviralexpression vector of the present invention can be used to infect bonemarrow cells which are subsequently transplanted into irradiatedrecipients. The pathology of the subject recipient is then examinedafter sufficient time has passed (e.g., six weeks).

To maximize recombinant protein expression in E. coli is to express theprotein in a host bacteria with an impaired capacity to proteolyticallycleave the recombinant protein (Gottesman (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.119-128). Another strategy is to alter the nucleic acid sequence of thenucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

The 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 expression vector can be a yeast expression vector, a vectorfor expression in insect cells, e.g., a baculovirus expression vector ora vector suitable for expression in mammalian cells.

When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Non-limiting examples of suitabletissue-specific promoters include the albumin promoter (liver-specific;Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters(Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particularpromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740;Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al.(1985) Science 230:912-916), and mammary gland-specific promoters (e.g.,milk whey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example, the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. Regulatory sequences (e.g., viralpromoters and/or enhancers) operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus. For a discussion of the regulation of gene expressionusing antisense genes see Weintraub et al., (1986) Reviews—Trends inGenetics 1:1.

Another aspect the invention provides a host cell which includes anucleic acid molecule described herein, e.g., a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 nucleic acidmolecule within a recombinant expression vector or a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 nucleicacid molecule containing sequences which allow it to homologouslyrecombine into a specific site of the host cell's genome. The terms“host cell” and “recombinant host cell” are used interchangeably herein.Such terms refer not only to the particular subject cell but to theprogeny or potential progeny of such a cell. Because certainmodifications can occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovary(CHO) cells or CV-1 origin, SV-40 (COS) cells). Other suitable hostcells are known to those skilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

A host cell of the invention can be used to produce (i.e., express) a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein. Accordingly, the invention further provides methods forproducing a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein using the host cells of the invention. Inone embodiment, the method includes culturing the host cell of theinvention (into which a recombinant expression vector encoding a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577protein has been introduced) in a suitable medium such that a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577protein is produced. In another embodiment, the method further includesisolating a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein from the medium or the host cell.

In another aspect, the invention features, a cell or purifiedpreparation of cells which include a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 transgene, or which otherwisemisexpress 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577. The cell preparation can consist of human ornon-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbitcells, or pig cells. In preferred embodiments, the cell or cells includea 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 transgene, e.g., a heterologous form of a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577, e.g., a genederived from humans (in the case of a non-human cell). The 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577transgene can be misexpressed, e.g., overexpressed or underexpressed. Inother preferred embodiments, the cell or cells include a gene whichmisexpresses an endogenous 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577, e.g., a gene the expression ofwhich is disrupted, e.g., a knockout. Such cells can serve as a modelfor studying disorders which are related to mutated or misexpressed27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 alleles or for use in drug screening.

In another aspect, the invention features, a human cell, e.g., ahematopoietic stem cell, transformed with nucleic acid which encodes asubject 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 polypeptide.

Also provided are cells, preferably human cells, e.g., humanhematopoietic or fibroblast cells, in which an endogenous 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 is underthe control of a regulatory sequence that does not normally control theexpression of the endogenous 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 gene. The expression characteristicsof an endogenous gene within a cell, e.g., a cell line or microorganism,can be modified by inserting a heterologous DNA regulatory element intothe genome of the cell such that the inserted regulatory element isoperably linked to the endogenous 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 gene. For example, anendogenous 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 gene which is “transcriptionally silent,” e.g.,not normally expressed, or expressed only at very low levels, can beactivated by inserting a regulatory element which is capable ofpromoting the expression of a normally expressed gene product in thatcell. Techniques such as targeted homologous recombinations, can be usedto insert the heterologous DNA as described in, e.g., Chappel, U.S. Pat.No. 5,272,071; WO 91/06667, published in May 16, 1991.

Transgenic Animals

The invention provides non-human transgenic animals. Such animals areuseful for studying the function and/or activity of a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteinand for identifying and/or evaluating modulators of 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 activity. Asused herein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians, and the like. A transgene is exogenous DNA or arearrangement, e.g., a deletion of endogenous chromosomal DNA, whichpreferably is integrated into or occurs in the genome of the cells of atransgenic animal. A transgene can direct the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal, other transgenes, e.g., a knockout, reduce expression. Thus, atransgenic animal can be one in which an endogenous 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene has beenaltered by, e.g., by homologous recombination between the endogenousgene and an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to atransgene of the invention to direct expression of a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteinto particular cells. A transgenic founder animal can be identified basedupon the presence of a 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 transgene in its genome and/or expressionof 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 mRNA in tissues or cells of the animals. A transgenic founderanimal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encoding a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein can further be bred to other transgenic animals carryingother transgenes.

27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 proteins or polypeptides can be expressed in transgenic animals orplants, e.g., a nucleic acid encoding the protein or polypeptide can beintroduced into the genome of an animal. In preferred embodiments thenucleic acid is placed under the control of a tissue specific promoter,e.g., a milk or egg specific promoter, and recovered from the milk oreggs produced by the animal. Suitable animals are mice, pigs, cows,goats, and sheep.

The invention also includes a population of cells from a transgenicanimal, as discussed, e.g., below.

Uses

The nucleic acid molecules, proteins, protein homologs, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic).

The isolated nucleic acid molecules of the invention can be used, forexample, to express a 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577mRNA (e.g., in a biological sample) or a genetic alteration in a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577gene, and to modulate 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 activity, as described further below. The27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 proteins can be used to treat disorders characterized byinsufficient or excessive production of a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 substrate orproduction of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 inhibitors. In addition, the 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteins can beused to screen for naturally occurring 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 substrates, to screenfor drugs or compounds which modulate 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 activity, as well as to treatdisorders characterized by insufficient or excessive production of27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein or production of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein forms which have decreased,aberrant or unwanted activity compared to 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 wild type protein(e.g., aberrant or deficient phospholipase activity, serinecarboxypeptidase activity, trypsin-like serine protease activity,aldehyde dehydrogenase activity, ubiquitin-protein ligase activity,protein kinase activity, hydrolase activity, matrix metalloproteinaseactivity, or other activity). Moreover, the anti-27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 antibodies ofthe invention can be used to detect and isolate 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteins,regulate the bioavailability of 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 proteins, and modulate 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577activity.

A method of evaluating a compound for the ability to interact with,e.g., bind, a subject 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 polypeptide is provided. The methodincludes: contacting the compound with the subject 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 polypeptide;and evaluating ability of the compound to interact with, e.g., to bindor form a complex with the subject 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 polypeptide. This method canbe performed in vitro, e.g., in a cell free system, or in vivo, e.g., ina two-hybrid interaction trap assay. This method can be used to identifynaturally occurring molecules which interact with subject 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577polypeptide. It can also be used to find natural or synthetic inhibitorsof subject 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 polypeptide. Screening methods are discussed inmore detail below.

Screening Assays:

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., proteins, peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which bind to 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteins, have astimulatory or inhibitory effect on, for example, 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 expression or27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 activity, or have a stimulatory or inhibitory effect on, forexample, the expression or activity of a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 substrate. Compoundsthus identified can be used to modulate the activity of target geneproducts (e.g., 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 genes) in a therapeutic protocol, to elaborate thebiological function of the target gene product, or to identify compoundsthat disrupt normal target gene interactions.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein or polypeptideor a biologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein or polypeptideor a biologically active portion thereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann et al. (1994)J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422-426; Zuckermann et al. (1994). J. Med. Chem. 37:2678-85; Cho etal. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233-51.

Libraries of compounds can be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein or biologically active portion thereof iscontacted with a test compound, and the ability of the test compound tomodulate 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 activity is determined. Determining the ability of thetest compound to modulate 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 activity can be accomplished bymonitoring, for example, phospholipase activity, serine carboxypeptidaseactivity, trypsin-like serine protease activity, aldehyde dehydrogenaseactivity, ubiquitin-protein ligase activity, protein kinase activity,hydrolase activity, matrix metalloproteinase activity, or otheractivity. The cell, for example, can be of mammalian origin, e.g.,human.

The ability of the test compound to modulate 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 binding to a compound,e.g., a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 substrate, or to bind to 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 can also be evaluated.This can be accomplished, for example, by coupling the compound, e.g.,the substrate, with a radioisotope or enzymatic label such that bindingof the compound, e.g., the substrate, to 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 can be determined bydetecting the labeled compound, e.g., substrate, in a complex.Alternatively, 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 could be coupled with a radioisotope or enzymaticlabel to monitor the ability of a test compound to modulate 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577binding to a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 substrate in a complex. For example, compounds(e.g., 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 substrates) can be labeled with ¹²⁵I, ¹⁴C, ³⁵S or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,compounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

The ability of a compound (e.g., a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 substrate) to interact with27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 with or without the labeling of any of the interactants can beevaluated. For example, a microphysiometer can be used to detect theinteraction of a compound with 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 without the labeling of either thecompound or the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577. McConnell et al. (1992) Science 257:1906-1912. Asused herein, a “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a compound and 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577.

In yet another embodiment, a cell-free assay is provided in which a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein or biologically active portion thereof is contacted with atest compound and the ability of the test compound to bind to the 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577protein or biologically active portion thereof is evaluated. Preferredbiologically active portions of the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 proteins to be used in assaysof the present invention include fragments which participate ininteractions with non-27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 molecules, e.g., fragments with highsurface probability scores.

Soluble and/or membrane-bound forms of isolated proteins (e.g., 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577proteins or biologically active portions thereof) can be used in thecell-free assays of the invention. When membrane-bound forms of theprotein are used, it may be desirable to utilize a solubilizing agent.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule can simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label can be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, determining the ability of the 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteinto bind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander andUrbaniczky (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr.Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA”detects biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcore). Changes in the mass at the bindingsurface (indicative of a binding event) result in alterations of therefractive index of light near the surface (the optical phenomenon ofsurface plasmon resonance (SPR)), resulting in a detectable signal whichcan be used as an indication of real-time reactions between biologicalmolecules.

In one embodiment, the target gene product or the test substance isanchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize either 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577, an anti-27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 antibodyor its target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein, or interaction of a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein with a targetmolecule in the presence and absence of a candidate compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577fusion proteins or glutathione-S-transferase/target fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtiter plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein, and the mixture incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH).

Following incubation, the beads or microtiter plate wells are washed toremove any unbound components, the matrix immobilized in the case ofbeads, complex determined either directly or indirectly, for example, asdescribed above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 binding or activity determined usingstandard techniques.

Other techniques for immobilizing either a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein or a targetmolecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein or target molecules can beprepared from biotin-NHS(N-hydroxy-succinimide) using techniques knownin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific or selective for the immobilizedcomponent (the antibody, in turn, can be directly labeled or indirectlylabeled with, e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactivewith 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 protein or target molecules but which do not interferewith binding of the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 protein to its target molecule. Suchantibodies can be derivatized to the wells of the plate, and unboundtarget or 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 protein trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein or targetmolecule, as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein or targetmolecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including but notlimited to: differential centrifugation (see, for example, Rivas andMinton (1993) Trends Biochem Sci 18:284-7); chromatography (gelfiltration chromatography, ion-exchange chromatography); electrophoresis(see, e.g., Ausubel et al., eds. (1999) Current Protocols in MolecularBiology, J. Wiley, New York.); and immunoprecipitation (see, forexample, Ausubel et al., eds. (1999) Current Protocols in MolecularBiology, J. Wiley, New York). Such resins and chromatographic techniquesare known to one skilled in the art (see, e.g., Heegaard (1998) J MolRecognit 11: 141-8; Hage and Tweed (1997) J Chromatogr B Biomed SciAppl. 699:499-525). Further, fluorescence energy transfer can also beconveniently utilized, as described herein, to detect binding withoutfurther purification of the complex from solution.

In a preferred embodiment, the assay includes contacting the 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577protein or biologically active portion thereof with a known compoundwhich binds 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein, wherein determining theability of the test compound to interact with a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteinincludes determining the ability of the test compound to preferentiallybind to 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 or biologically active portion thereof, or to modulatethe activity of a target molecule, as compared to the known compound.

The target gene products of the invention can, in vivo, interact withone or more cellular or extracellular macromolecules, such as proteins.For the purposes of this discussion, such cellular and extracellularmacromolecules are referred to herein as “binding partners.” Compoundsthat disrupt such interactions can be useful in regulating the activityof the target gene product. Such compounds can include, but are notlimited to molecules such as antibodies, peptides, and small molecules.The preferred target genes/products for use in this embodiment are the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 genes herein identified. In an alternative embodiment, theinvention provides methods for determining the ability of the testcompound to modulate the activity of a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein throughmodulation of the activity of a downstream effector of a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552, 65577 or 56919target molecule. For example, the activity of the effector molecule onan appropriate target can be determined, or the binding of the effectorto an appropriate target can be determined, as previously described.

To identify compounds that interfere with the interaction between thetarget gene product and its cellular or extracellular bindingpartner(s), a reaction mixture containing the target gene product andthe binding partner is prepared, under conditions and for a timesufficient, to allow the two products to form complex. In order to testan inhibitory agent, the reaction mixture is provided in the presenceand absence of the test compound. The test compound can be initiallyincluded in the reaction mixture, or can be added at a time subsequentto the addition of the target gene and its cellular or extracellularbinding partner. Control reaction mixtures are incubated without thetest compound or with a placebo. The formation of any complexes betweenthe target gene product and the cellular or extracellular bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction of thetarget gene product and the interactive binding partner.

Additionally, complex formation within reaction mixtures containing thetest compound and normal target gene product can also be compared tocomplex formation within reaction mixtures containing the test compoundand mutant target gene product. This comparison can be important inthose cases wherein it is desirable to identify compounds that disruptinteractions of mutant but not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format.Heterogeneous assays involve anchoring either the target gene product orthe binding partner onto a solid phase, and detecting complexes anchoredon the solid phase at the end of the reaction. In homogeneous assays,the entire reaction is carried out in a liquid phase. In eitherapproach, the order of addition of reactants can be varied to obtaindifferent information about the compounds being tested. For example,test compounds that interfere with the interaction between the targetgene products and the binding partners, e.g., by competition, can beidentified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

In a heterogeneous assay system, either the target gene product or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface (e.g., a microtiter plate), while the non-anchoredspecies is labeled, either directly or indirectly. The anchored speciescan be immobilized by non-covalent or covalent attachments.Alternatively, an immobilized antibody specific or selective for thespecies to be anchored can be used to anchor the species to the solidsurface.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific or selective for the initiallynon-immobilized species (the antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody). Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific or selective for one of the bindingcomponents to anchor any complexes formed in solution, and a labeledantibody specific or selective for the other partner to detect anchoredcomplexes. Again, depending upon the order of addition of reactants tothe liquid phase, test compounds that inhibit complex or that disruptpreformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. For example, a preformed complex of the target gene product andthe interactive cellular or extracellular binding partner product isprepared in that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 thatutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances that disrupt target geneproduct-binding partner interaction can be identified.

In yet another aspect, the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 proteins can be used as “baitproteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S.Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 (“27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577-binding proteins” or “27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577-bp”) and areinvolved in 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 activity. Such 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577-bps can be activators orinhibitors of signals by the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 proteins or 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552, 65577 or 56919 targetsas, for example, downstream elements of a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577-mediated signalingpathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. (Alternatively the:27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein can be the fused to the activator domain.) If the “bait”and the “prey” proteins are able to interact, in vivo, forming a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., lacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein.

In another embodiment, modulators of 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 expression are identified.For example, a cell or cell free mixture is contacted with a candidatecompound and the expression of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 mRNA or protein evaluated relativeto the level of expression of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 mRNA or protein in the absence ofthe candidate compound. When expression of 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 mRNA or protein isgreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 mRNA orprotein expression. Alternatively, when expression of 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 mRNA orprotein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 mRNA or protein expression. Thelevel of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 mRNA or protein expression can be determined by methodsdescribed herein for detecting 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein can beconfirmed in vivo, e.g., in an animal such as an animal model foraberrant or deficient phospholipase activity, serine carboxypeptidaseactivity, trypsin-like serine protease activity, aldehyde dehydrogenaseactivity, ubiquitin-protein ligase activity, protein kinase activity,hydrolase activity or matrix metalloproteinase activity.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 modulating agent, an antisense 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 nucleic acidmolecule, a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577-specific antibody, or a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577-bindingpartner) in an appropriate animal model to determine the efficacy,toxicity, side effects, or mechanism of action, of treatment with suchan agent. Furthermore, novel agents identified by the above-describedscreening assays can be used for treatments as described herein.

Detection Assays

Portions or fragments of the nucleic acid sequences identified hereincan be used as polynucleotide reagents. For example, these sequences canbe used to: (i) map their respective genes on a chromosome e.g., tolocate gene regions associated with genetic disease or to associate27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 with a disease; (ii) identify an individual from a minutebiological sample (tissue typing); and (iii) aid in forensicidentification of a biological sample. These applications are describedin the subsections below.

Chromosome Mapping

The 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 nucleotide sequences or portions thereof can be used to map thelocation of the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 genes on a chromosome. This process is calledchromosome mapping. Chromosome mapping is useful in correlating the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 sequences with genes associated with disease.

Briefly, 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 nucleotidesequences. These primers can then be used for PCR screening of somaticcell hybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to the 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 sequences willyield an amplified fragment.

A panel of somatic cell hybrids in which each cell line contains eithera single human chromosome or a small number of human chromosomes, and afull set of mouse chromosomes, can allow easy mapping of individualgenes to specific human chromosomes. (D'Eustachio et al. (1983) Science220:919-924).

Other mapping strategies e.g., in situ hybridization (described in Fanet al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screeningwith labeled flow-sorted chromosomes, and pre-selection by hybridizationto chromosome specific cDNA libraries can be used to map 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 to achromosomal location.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. The FISH technique can be used with aDNA sequence as short as 500 or 600 bases. However, clones larger than1,000 bases have a higher likelihood of binding to a unique chromosomallocation with sufficient signal intensity for simple detection.Preferably 1,000 bases, and more preferably 2,000 bases will suffice toget good results at a reasonable amount of time. For a review of thistechnique, see Verma et al. (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in McKusick,Mendelian Inheritance in Man, available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between a gene and adisease, mapped to the same chromosomal region, can then be identifiedthrough linkage analysis (co-inheritance of physically adjacent genes),described in, for example, Egeland et al. (1987) Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

Tissue Typing

27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 sequences can be used to identify individuals from biologicalsamples using, e.g., restriction fragment length polymorphism (RFLP). Inthis technique, an individual's genomic DNA is digested with one or morerestriction enzymes, the fragments separated, e.g., in a Southern blot,and probed to yield bands for identification. The sequences of thepresent invention are useful as additional DNA markers for RFLP(described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can also be used todetermine the actual base-by-base DNA sequence of selected portions ofan individual's genome. Thus, the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 nucleotide sequencesdescribed herein can be used to prepare two PCR primers from the 5′ and3′ ends of the sequences. These primers can then be used to amplify anindividual's DNA and subsequently sequence it. Panels of correspondingDNA sequences from individuals, prepared in this manner, can provideunique individual identifications, as each individual will have a uniqueset of such DNA sequences due to allelic differences.

Allelic variation occurs to some degree in the coding regions of thesesequences, and to a greater degree in the noncoding regions. Each of thesequences described herein can, to some degree, be used as a standardagainst which DNA from an individual can be compared for identificationpurposes. Because greater numbers of polymorphisms occur in thenoncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1, 4, 11, 20, 25, 29,35, 38, 41, 73, 76 or 86 can provide positive individual identificationwith a panel of perhaps 10 to 1,000 primers which each yield a noncodingamplified sequence of 100 bases. If predicted coding sequences, such asthose in SEQ ID NO: 3, 6, 13, 22, 27, 31, 37, 40, 43, 75, 78 or 88 areused, a more appropriate number of primers for positive individualidentification would be 500-2,000.

If a panel of reagents from 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 nucleotide sequences describedherein is used to generate a unique identification database for anindividual, those same reagents can later be used to identify tissuefrom that individual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

Use of Partial 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. To make such an identification, PCR technology can be used toamplify DNA sequences taken from very small biological samples such astissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, orsemen found at a crime scene. The amplified sequence can then becompared to a standard, thereby allowing identification of the origin ofthe biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1, 4, 11, 20, 25, 29, 35, 38,41, 73, 76 or 86 (e.g., fragments derived from the noncoding regions ofSEQ ID NO: 1, 4, 11, 20, 25, 29, 35, 38, 41, 73, 76 or 86 having alength of at least 20 bases, preferably at least 30 bases) areparticularly appropriate for this use.

The 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue. This can be very useful in cases where aforensic pathologist is presented with a tissue of unknown origin.Panels of such 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 probes can be used to identify tissue by speciesand/or by organ type.

In a similar fashion, these reagents, e.g., 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 primers or probes canbe used to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual.

Generally, the invention provides, a method of determining if a subjectis at risk for a disorder related to a lesion in or the misexpression ofa gene which encodes 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577.

Such disorders include, e.g., a disorder associated with themisexpression of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 gene; a cellular proliferative and/ordifferentiative disorder, brain, blood vessel, platelet, breast, colon,kidney, lung, ovarian, prostate, hematopoeitic, pancreatic, skeletalmuscle, testicular, skin, hormonal, associated with bone metabolism,immune e.g., inflammatory, cardiovascular, endothelial cell, liver,viral diseases, pain, metabolic, anemias, angiogenesis, neoplastic,endocrine, neurological or heart disorder.

The method includes one or more of the following: detecting, in a tissueof the subject, the presence or absence of a mutation which affects theexpression of the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 gene, or detecting the presence or absenceof a mutation in a region which controls the expression of the gene,e.g., a mutation in the 5′ control region; detecting, in a tissue of thesubject, the presence or absence of a mutation which alters thestructure of the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 gene; detecting, in a tissue of the subject, themisexpression of the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 gene, at the mRNA level, e.g., detecting anon-wild type level of an mRNA; or detecting, in a tissue of thesubject, the misexpression of the gene, at the protein level, e.g.,detecting a non-wild type level of a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 polypeptide.

In preferred embodiments the method includes: ascertaining the existenceof at least one of: a deletion of one or more nucleotides from the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 gene; an insertion of one or more nucleotides into the gene, apoint mutation, e.g., a substitution of one or more nucleotides of thegene, a gross chromosomal rearrangement of the gene, e.g., atranslocation, inversion, or deletion.

For example, detecting the genetic lesion can include: (i) providing aprobe/primer including an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequencefrom SEQ ID NO:1, 4, 11, 20, 25, 29, 35, 38, 41, 73, 76 or 86, ornaturally occurring mutants thereof or 5′ or 3′ flanking sequencesnaturally associated with the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 gene; (ii) exposing the probe/primerto nucleic acid of the tissue; and detecting, by hybridization, e.g., insitu hybridization, of the probe/primer to the nucleic acid, thepresence or absence of the genetic lesion.

In preferred embodiments detecting the misexpression includesascertaining the existence of at least one of: an alteration in thelevel of a messenger RNA transcript of the 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene; the presence ofa non-wild type splicing pattern of a messenger RNA transcript of thegene; or a non-wild type level of 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577.

Methods of the invention can be used prenatally or to determine if asubject's offspring will be at risk for a disorder.

In preferred embodiments the method includes determining the structureof a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 gene, an abnormal structure being indicative of risk forthe disorder.

In preferred embodiments the method includes contacting a sample fromthe subject with an antibody to the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein or a nucleic acid,which hybridizes specifically with the gene. These and other embodimentsare discussed below.

Diagnostic and Prognostic Assays

The presence, level, or absence of 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein or nucleic acid in abiological sample can be evaluated by obtaining a biological sample froma test subject and contacting the biological sample with a compound oran agent capable of detecting 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein or nucleic acid (e.g., mRNA,genomic DNA) that encodes 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein such that the presence of27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein or nucleic acid is detected in the biological sample. Theterm “biological sample” includes tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. A preferred biological sample is serum. The level ofexpression of the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 gene can be measured in a number of ways,including, but not limited to: measuring the mRNA encoded by the 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577genes; measuring the amount of protein encoded by the 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 genes;or measuring the activity of the protein encoded by the 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 genes.

The level of mRNA corresponding to the 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene in a cell can bedetermined both by in situ and by in vitro formats.

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 nucleic acid,such as the nucleic acid of SEQ ID NO: 1, 4, 11, 20, 25, 29, 35, 38, 41,73, 76 or 86, or a portion thereof, such as an oligonucleotide of atleast 7, 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 mRNA or genomic DNA. Other suitable probes for use in thediagnostic assays are described herein.

In one format, mRNA (or cDNA) is immobilized on a surface and contactedwith the probes, for example by running the isolated mRNA on an agarosegel and transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probes are immobilized ona surface and the mRNA (or cDNA) is contacted with the probes, forexample, in a two-dimensional gene chip array. A skilled artisan canadapt known mRNA detection methods for use in detecting the level ofmRNA encoded by the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 genes.

The level of mRNA in a sample that is encoded by one of 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 can beevaluated with nucleic acid amplification, e.g., by rtPCR (Mullis (1987)U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc.Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication(Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878),transcriptional amplification system (Kwoh et al., (1989), Proc. Natl.Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988)Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S.Pat. No. 5,854,033) or any other nucleic acid amplification method,followed by the detection of the amplified molecules using techniquesknown in the art. As used herein, amplification primers are defined asbeing a pair of nucleic acid molecules that can anneal to 5′ or 3′regions of a gene (plus and minus strands, respectively, or vice-versa)and contain a short region in between. In general, amplification primersare from about 10 to 30 nucleotides in length and flank a region fromabout 50 to 200 nucleotides in length. Under appropriate conditions andwith appropriate reagents, such primers permit the amplification of anucleic acid molecule comprising the nucleotide sequence flanked by theprimers.

For in situ methods, a cell or tissue sample can be prepared/processedand immobilized on a support, typically a glass slide, and thencontacted with a probe that can hybridize to mRNA that encodes the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 gene being analyzed.

In another embodiment, the methods further contacting a control samplewith a compound or agent capable of detecting 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 mRNA, orgenomic DNA, and comparing the presence of 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 mRNA or genomic DNA inthe control sample with the presence of 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 mRNA or genomic DNA inthe test sample.

A variety of methods can be used to determine the level of proteinencoded by 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577. In general, these methods include contacting anagent that selectively binds to the protein, such as an antibody with asample, to evaluate the level of protein in the sample. In a preferredembodiment, the antibody bears a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with a detectablesubstance. Examples of detectable substances are provided herein.

The detection methods can be used to detect 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein in abiological sample in vitro as well as in vivo. In vitro techniques fordetection of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein include enzyme linked immunosorbent assays(ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay(EIA), radioimmunoassay (RIA), and Western blot analysis. In vivotechniques for detection of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein include introducing into asubject a labeled anti-27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

In another embodiment, the methods further include contacting thecontrol sample with a compound or agent capable of detecting 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577protein, and comparing the presence of 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein in the controlsample with the presence of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein in the test sample.

The invention also includes kits for detecting the presence of 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577in a biological sample. For example, the kit can include a compound oragent capable of detecting 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein or mRNA in a biologicalsample; and a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 protein or nucleic acid.

For antibody-based kits, the kit can include: (1) a first antibody(e.g., attached to a solid support) which binds to a polypeptidecorresponding to a marker of the invention; and, optionally, (2) asecond, different antibody which binds to either the polypeptide or thefirst antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can include: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also includes a buffering agent, apreservative, or a protein stabilizing agent. The kit can also includescomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

The diagnostic methods described herein can identify subjects having, orat risk of developing, a disease or disorder associated withmisexpressed or aberrant or unwanted 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 expression or activity. Asused herein, the term “unwanted” includes an unwanted phenomenoninvolved in a biological response such as pain or deregulated cellproliferation.

In one embodiment, a disease or disorder associated with aberrant orunwanted 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 expression or activity is identified. A test sample isobtained from a subject and 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 protein or nucleic acid (e.g., mRNAor genomic DNA) is evaluated, wherein the level, e.g., the presence orabsence, of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein or nucleic acid is diagnostic for asubject having or at risk of developing a disease or disorder associatedwith aberrant or unwanted 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 expression or activity. As usedherein, a “test sample” refers to a biological sample obtained from asubject of interest, including a biological fluid (e.g., serum), cellsample, or tissue.

The prognostic assays described herein can be used to determine whethera subject can be administered an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) to treat a disease or disorder associated with aberrantor unwanted 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 expression or activity. For example, such methodscan be used to determine whether a subject can be effectively treatedwith an agent for a cellular proliferative and/or differentiativedisorder, brain, blood vessel, platelet, breast, colon, kidney, lung,ovarian, prostate, hematopoeitic, pancreatic, skeletal muscle,testicular, skin, hormonal, associated with bone metabolism, immunee.g., inflammatory, cardiovascular, endothelial cell, liver, viraldiseases, pain, metabolic, anemias, angiogenesis, neoplastic, endocrine,neurological or heart disorder.

The methods of the invention can also be used to detect geneticalterations in a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation in27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein activity or nucleic acid expression, such as a cellularproliferative and/or differentiative disorder, brain, blood vessel,platelet, breast, colon, kidney, lung, ovarian, prostate, hematopoeitic,pancreatic, skeletal muscle, testicular, skin, hormonal, associated withbone metabolism, immune e.g., inflammatory, cardiovascular, endothelialcell, liver, viral diseases, pain, metabolic, anemias, angiogenesis,neoplastic, endocrine, neurological or heart disorder. In preferredembodiments, the methods include detecting, in a sample from thesubject, the presence or absence of a genetic alteration characterizedby at least one of an alteration affecting the integrity of a geneencoding a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577-protein, or the mis-expression of the 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577gene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 gene; 2) an addition of one or morenucleotides to a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 gene; 3) a substitution of one or more nucleotidesof a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 gene, 4) a chromosomal rearrangement of a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene; 5)an alteration in the level of a messenger RNA transcript of a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577gene, 6) aberrant modification of a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 gene, such as of themethylation pattern of the genomic DNA, 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene, 8)a non-wild type level of a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577-protein, 9) allelic loss of a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577gene, and 10) inappropriate post-translational modification of a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577-protein.

An alteration can be detected without a probe/primer in a polymerasechain reaction, such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR), the latter of which can be particularlyuseful for detecting point mutations in the 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577-gene. This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the sample,contacting the nucleic acid sample with one or more primers whichspecifically hybridize to a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 gene under conditions such thathybridization and amplification of the 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene (if present)occurs, and detecting the presence or absence of an amplificationproduct, or detecting the size of the amplification product andcomparing the length to a control sample. It is anticipated that PCRand/or LCR may be desirable to use as a preliminary amplification stepin conjunction with any of the techniques used for detecting mutationsdescribed herein. Alternatively, other amplification methods describedherein or known in the art can be used.

In another embodiment, mutations in a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 gene from a sample cell canbe identified by detecting alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined, e.g., by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, twodimensional arrays, e.g., chip based arrays. Such arrays include aplurality of addresses, each of which is positionally distinguishablefrom the other. A different probe is located at each address of theplurality. The arrays can have a high density of addresses, e.g., cancontain hundreds or thousands of oligonucleotides probes (Cronin et al.(1996) Human Mutation 7: 244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For example, genetic mutations in 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 can be identified intwo dimensional arrays containing light-generated DNA probes asdescribed in Cronin, M. T. et al. supra. Briefly, a first hybridizationarray of probes can be used to scan through long stretches of DNA in asample and control to identify base changes between the sequences bymaking linear arrays of sequential overlapping probes. This step allowsthe identification of point mutations. This step is followed by a secondhybridization array that allows the characterization of specificmutations by using smaller, specialized probe arrays complementary toall variants or mutations detected. Each mutation array is composed ofparallel probe sets, one complementary to the wild-type gene and theother complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene anddetect mutations by comparing the sequence of the sample 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 with thecorresponding wild-type (control) sequence. Automated sequencingprocedures can be utilized when performing the diagnostic assays (Naeveet al. (1995) Biotechniques 19:448-53), including sequencing by massspectrometry.

Other methods for detecting mutations in the 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242; Cotton et al. (1988) Proc. Natl. Acad Sci USA 85:4397; Saleebaet al. (1992) Methods Enzymol. 217:286-295).

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S.Pat. No. 5,459,039).

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 genes. For example, single strandconformation polymorphism (SSCP) can be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton(1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech.Appl. 9:73-79). Single-stranded DNA fragments of sample and control27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 nucleic acids will be denatured and allowed to renature. Thesecondary structure of single-stranded nucleic acids varies according tosequence, the resulting alteration in electrophoretic mobility enablesthe detection of even a single base change. The DNA fragments can belabeled or detected with labeled probes. The sensitivity of the assaycan be enhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension (Saiki et al. (1986) Nature324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci USA 86:6230).

Alternatively, allele specific amplification technology which depends onselective PCR amplification can be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationcan carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification can also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189-93). Insuch cases, ligation will occur only if there is a perfect match at the3′ end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein can be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which can be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene.

Use of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 Molecules as Surrogate Markers

The 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 molecules of the invention are also useful as markers ofdisorders or disease states, as markers for precursors of diseasestates, as markers for predisposition of disease states, as markers ofdrug activity, or as markers of the pharmacogenomic profile of asubject. Using the methods described herein, the presence, absenceand/or quantity of the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 molecules of the invention can be detected,and can be correlated with one or more biological states in vivo. Forexample, the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 molecules of the invention can serve as surrogatemarkers for one or more disorders or disease states or for conditionsleading up to disease states. As used herein, a “surrogate marker” is anobjective biochemical marker which correlates with the absence orpresence of a disease or disorder, or with the progression of a diseaseor disorder (e.g., with the presence or absence of a tumor). Thepresence or quantity of such markers is independent of the disease.Therefore, these markers can serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies (e.g., early stage tumors), or when an assessmentof disease progression is desired before a potentially dangerousclinical endpoint is reached (e.g., an assessment of cardiovasculardisease can be made using cholesterol levels as a surrogate marker, andan analysis of HIV infection can be made using HIV RNA levels as asurrogate marker, well in advance of the undesirable clinical outcomesof myocardial infarction or fully-developed AIDS). Examples of the useof surrogate markers in the art include: Koomen et al. (2000) J. Mass.Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

The 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 molecules of the invention are also useful as pharmacodynamicmarkers. As used herein, a “pharmacodynamic marker” is an objectivebiochemical marker which correlates specifically with drug effects. Thepresence or quantity of a pharmacodynamic marker is not related to thedisease state or disorder for which the drug is being administered;therefore, the presence or quantity of the marker is indicative of thepresence or activity of the drug in a subject. For example, apharmacodynamic marker can be indicative of the concentration of thedrug in a biological tissue, in that the marker is either expressed ortranscribed or not expressed or transcribed in that tissue inrelationship to the level of the drug. In this fashion, the distributionor uptake of the drug can be monitored by the pharmacodynamic marker.Similarly, the presence or quantity of the pharmacodynamic marker can berelated to the presence or quantity of the metabolic product of a drug,such that the presence or quantity of the marker is indicative of therelative breakdown rate of the drug in vivo. Pharmacodynamic markers areof particular use in increasing the sensitivity of detection of drugeffects, particularly when the drug is administered in low doses. Sinceeven a small amount of a drug can be sufficient to activate multiplerounds of marker (e.g., a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 marker) transcription or expression,the amplified marker can be in a quantity which is more readilydetectable than the drug itself. Also, the marker can be more easilydetected due to the nature of the marker itself; for example, using themethods described herein, anti-27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 antibodies can be employed in animmune-based detection system for a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein marker, or 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577-specific radiolabeled probes can be used to detect a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 mRNAmarker. Furthermore, the use of a pharmacodynamic marker can offermechanism-based prediction of risk due to drug treatment beyond therange of possible direct observations. Examples of the use ofpharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No.6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238;Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; andNicolau (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

The 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 molecules of the invention are also useful as pharmacogenomicmarkers. As used herein, a “pharmacogenomic marker” is an objectivebiochemical marker which correlates with a specific clinical drugresponse or susceptibility in a subject (see, e.g., McLeod et al. (1999)Eur. J. Cancer 35:1650-1652). The presence or quantity of thepharmacogenomic marker is related to the predicted response of thesubject to a specific drug or class of drugs prior to administration ofthe drug. By assessing the presence or quantity of one or morepharmacogenomic markers in a subject, a drug therapy which is mostappropriate for the subject, or which is predicted to have a greaterdegree of success, can be selected. For example, based on the presenceor quantity of RNA, or protein (e.g., 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 protein or RNA) for specifictumor markers in a subject, a drug or course of treatment can beselected that is optimized for the treatment of the specific tumorlikely to be present in the subject. Similarly, the presence or absenceof a specific sequence mutation in 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 DNA can correlate with a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 drug response. The use of pharmacogenomic markers thereforepermits the application of the most appropriate treatment for eachsubject without having to administer the therapy.

Pharmaceutical Compositions

The nucleic acid and polypeptides, fragments thereof, as well asanti-27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 antibodies (also referred to herein as “activecompounds”) of the invention can be incorporated into pharmaceuticalcompositions. Such compositions typically include the nucleic acidmolecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” includes solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors can influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide, or antibody, unconjugated or conjugated asdescribed herein, can include a single treatment or, preferably, caninclude a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight(generally 10 mg/kg to 20 mg/kg). If the antibody is to act in thebrain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

The present invention encompasses agents which modulate expression oractivity. An agent can, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per, kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher can, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577expression or activity. As used herein, the term “treatment” is definedas the application or administration of a therapeutic agent to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. A therapeutic agent includes, but is notlimited to, small molecules, peptides, antibodies, ribozymes andantisense oligonucleotides.

With regards to both prophylactic and therapeutic methods of treatment,such treatments can be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”.) Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577molecules of the present invention or 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 modulators according to thatindividual's drug response genotype. Pharmacogenomics allows a clinicianor physician to target prophylactic or therapeutic treatments topatients who will most benefit from the treatment and to avoid treatmentof patients who will experience toxic drug-related side effects.

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwanted27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 expression or activity, by administering to the subject a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577or an agent which modulates 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 expression or at least one 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577activity. Subjects at risk for a disease which is caused or contributedto by aberrant or unwanted 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 aberrance, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type of27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 aberrance, for example, a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577, 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 agonist or 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577antagonist agent can be used for treating the subject. The appropriateagent can be determined based on screening assays described herein.

It is possible that some 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 disorders can be caused, at least inpart, by an abnormal level of gene product, or by the presence of a geneproduct exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of such gene products would bring about theamelioration of disorder symptoms.

The 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 molecules can act as novel diagnostic targets and therapeuticagents for controlling one or more of a cellular proliferative and/ordifferentiative disorder, brain, blood vessel, platelet, breast, colon,kidney, lung, ovarian, prostate, hematopoeitic, pancreatic, skeletalmuscle, testicular, skin, hormonal, associated with bone metabolism,immune e.g., inflammatory, cardiovascular, endothelial cell, liver,viral diseases, pain, metabolic, anemias, angiogenesis, neoplastic,endocrine, neurological or heart disorder, all of which are describedabove.

As discussed, successful treatment of 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 disorders can be broughtabout by techniques that serve to inhibit the expression or activity oftarget gene products. For example, compounds, e.g., an agent identifiedusing an assays described above, that proves to exhibit negativemodulatory activity, can be used in accordance with the invention toprevent and/or ameliorate symptoms of 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 disorders. Such molecules caninclude, but are not limited to peptides, phosphopeptides, small organicor inorganic molecules, or antibodies (including, for example,polyclonal, monoclonal, humanized, human, anti-idiotypic, chimeric orsingle chain antibodies, and Fab, F(ab′)₂ and Fab expression libraryfragments, scFV molecules, and epitope-binding fragments thereof).

Further, antisense and ribozyme molecules that inhibit expression of thetarget gene can also be used in accordance with the invention to reducethe level of target gene expression, thus effectively reducing the levelof target gene activity. Still further, triple helix molecules can beutilized in reducing the level of target gene activity. Antisense,ribozyme and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helixmolecules to reduce or inhibit mutant gene expression can also reduce orinhibit the transcription (triple helix) and/or translation (antisense,ribozyme) of mRNA produced by normal target gene alleles, such that theconcentration of normal target gene product present can be lower than isnecessary for a normal phenotype. In such cases, nucleic acid moleculesthat encode and express target gene polypeptides exhibiting normaltarget gene activity can be introduced into cells via gene therapymethod. Alternatively, in instances in that the target gene encodes anextracellular protein, it can be preferable to co-administer normaltarget gene protein into the cell or tissue in order to maintain therequisite level of cellular or tissue target gene activity.

Another method by which nucleic acid molecules can be utilized intreating or preventing a disease characterized by 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 expression isthrough the use of aptamer molecules specific for 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein.Aptamers are nucleic acid molecules having a tertiary structure whichpermits them to specifically or selectively bind to protein ligands(see, e.g., Osborne et al. (1997) Curr. Opin. Chem Biol. 1: 5-9; andPatel (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid moleculescan in many cases be more conveniently introduced into target cells thantherapeutic protein molecules can be, aptamers offer a method by which27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 protein activity can be specifically decreased without theintroduction of drugs or other molecules which can have pluripotenteffects.

Antibodies can be generated that are both specific for target geneproduct and that reduce target gene product activity. Such antibodiescan, therefore, by administered in instances whereby negative modulatorytechniques are appropriate for the treatment of 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 disorders. Fora description of antibodies, see the Antibody section above.

In circumstances wherein injection of an animal or a human subject witha 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 protein or epitope for stimulating antibody production isharmful to the subject, it is possible to generate an immune responseagainst 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 through the use of anti-idiotypic antibodies (see, forexample, Herlyn (1999) Ann Med 31:66-78; and Bhattacharya-Chatterjee andFoon (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibodyis introduced into a mammal or human subject, it should stimulate theproduction of anti-anti-idiotypic antibodies, which should be specificto the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 protein.

Vaccines directed to a disease characterized by 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 expression canalso be generated in this fashion.

In instances where the target antigen is intracellular and wholeantibodies are used, internalizing antibodies can be preferred.Lipofectin or liposomes can be used to deliver the antibody or afragment of the Fab region that binds to the target antigen into cells.Where fragments of the antibody are used, the smallest inhibitoryfragment that binds to the target antigen is preferred. For example,peptides having an amino acid sequence corresponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizingantibodies that bind to intracellular target antigens can also beadministered. Such single chain antibodies can be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population (see e.g., Marasco et al.(1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

The identified compounds that inhibit target gene expression, synthesisand/or activity can be administered to a patient at therapeuticallyeffective doses to prevent, treat or ameliorate 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 disorders. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of the disorders.Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures as described above.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

Another example of determination of effective dose for an individual isthe ability to directly assay levels of “free” and “bound” compound inthe serum of the test subject. Such assays can utilize antibody mimicsand/or “biosensors” that have been created through molecular imprintingtechniques. The compound which is able to modulate 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 activity isused as a template, or “imprinting molecule”, to spatially organizepolymerizable monomers prior to their polymerization with catalyticreagents. The subsequent removal of the imprinted molecule leaves apolymer matrix which contains a repeated “negative image” of thecompound and is able to selectively rebind the molecule under biologicalassay conditions. A detailed review of this technique can be seen inAnsell et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea(1994) Trends in Polymer Science 2:166-173. Such “imprinted” affinitymatrixes are amenable to ligand-binding assays, whereby the immobilizedmonoclonal antibody component is replaced by an appropriately imprintedmatrix. An example of the use of such matrixes in this way can be seenin Vlatakis et al (1993) Nature 361:645-647. Through the use ofisotope-labeling, the “free” concentration of compound which modulatesthe expression or activity of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 can be readily monitored and used incalculations of IC₅₀.

Such “imprinted” affinity matrixes can also be designed to includefluorescent groups whose photon-emitting properties measurably changeupon local and selective binding of target compound. These changes canbe readily assayed in real time using appropriate fiberoptic devices, inturn allowing the dose in a test subject to be quickly optimized basedon its individual IC₅₀. An rudimentary example of such a “biosensor” isdiscussed in Kriz et al (1995) Analytical Chemistry 67:2142-2144.

Another aspect of the invention pertains to methods of modulating 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577expression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 or agent that modulates one or moreof the activities of 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 protein activity associated with the cell.An agent that modulates 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 protein activity can be an agent asdescribed herein, such as a nucleic acid or a protein, anaturally-occurring target molecule of a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein (e.g., a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 substrate or receptor), a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 antibody, a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 agonistor antagonist, a peptidomimetic of a 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 agonist or antagonist, orother small molecule.

In one embodiment, the agent stimulates one or 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 activities.Examples of such stimulatory agents include active 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein and anucleic acid molecule encoding 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577. In another embodiment, the agentinhibits one or more 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 activities. Examples of such inhibitoryagents include antisense 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 nucleic acid molecules, anti-27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577antibodies, and 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 inhibitors. These modulatory methods can beperformed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein ornucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g., upregulates or down regulates) 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 expression or activity. In anotherembodiment, the method involves administering a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 protein ornucleic acid molecule as therapy to compensate for reduced, aberrant, orunwanted 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 expression or activity.

Stimulation of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 activity is desirable in situations in which27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 is abnormally down-regulated and/or in which increased 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577activity is likely to have a beneficial effect. For example, stimulationof 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 activity is desirable in situations in which a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 isdownregulated and/or in which increased 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 activity is likely tohave a beneficial effect. Likewise, inhibition of 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 activity isdesirable in situations in which 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 is abnormally upregulatedand/or in which decreased 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 activity is likely to have abeneficial effect.

Pharmacogenomics

The 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577activity (e.g., 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577-associated disorders (e.g.,aberrant or deficient phospholipase activity, serine carboxypeptidaseactivity, trypsin-like serine protease activity, aldehyde dehydrogenaseactivity, ubiquitin-protein ligase activity, protein kinase activity,hydrolase activity or matrix metalloproteinase activity.) associatedwith aberrant or unwanted 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 activity.

In conjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) can be considered. Differencesin metabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician canconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 molecule or27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 modulator as well as tailoring the dosage and/or therapeuticregimen of treatment with a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 molecule or 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum et al. (1996)Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder et al. (1997) Clin.Chem. 43:254-266. In general, two types of pharmacogenetic conditionscan be differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare genetic defects or as naturally-occurringpolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(anti-malarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP can occur once per every 1000 bases of DNA. ASNP can be involved in a disease process, however, the vast majority cannot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that can becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577protein of the present invention), all common variants of that gene canbe fairly easily identified in the population and it can be determinedif having one version of the gene versus another is associated with aparticular drug response.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 moleculeor 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 modulator of the present invention) can give an indicationwhether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 moleculeor 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 modulator, such as a modulator identified by one of theexemplary screening assays described herein.

The present invention further provides methods for identifying newagents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 genes of the present invention, wherein theseproducts can be associated with resistance of the cells to a therapeuticagent. Specifically, the activity of the proteins encoded by the 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577genes of the present invention can be used as a basis for identifyingagents for overcoming agent resistance. By blocking the activity of oneor more of the resistance proteins, target cells, e.g., human cells,will become sensitive to treatment with an agent to which the unmodifiedtarget cells were resistant.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 protein can be applied in clinical trials. Forexample, the effectiveness of an agent determined by a screening assayas described herein to increase 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 gene expression, proteinlevels, or upregulate 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 activity, can be monitored in clinicaltrials of subjects exhibiting decreased 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene expression,protein levels, or downregulated 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 activity. Alternatively, theeffectiveness of an agent determined by a screening assay to decrease27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 gene expression, protein levels, or down-regulate 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577activity, can be monitored in clinical trials of subjects exhibitingincreased 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 gene expression, protein levels, or upregulated 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577activity. In such clinical trials, the expression or activity of a27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 gene, and preferably, other genes that have been implicated in,for example, a protein kinase-associated or another 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577-associateddisorder can be used as a “read out” or markers of the phenotype of aparticular cell.

OTHER EMBODIMENTS

In another aspect, the invention features a method of analyzing aplurality of capture probes. The method is useful, e.g., to analyze geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence, wherein the capture probes are from acell or subject which expresses 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 or from a cell or subject inwhich a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 mediated response has been elicited; contacting the arraywith a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 nucleic acid (preferably purified), a 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577polypeptide (preferably purified), or an anti-27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 antibody, andthereby evaluating the plurality of capture probes. Binding, e.g., inthe case of a nucleic acid, hybridization with a capture probe at anaddress of the plurality, is detected, e.g., by a signal generated froma label attached to the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 nucleic acid, polypeptide, or antibody.

The capture probes can be a set of nucleic acids from a selected sample,e.g., a sample of nucleic acids derived from a control or non-stimulatedtissue or cell.

The method can include contacting the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 nucleic acid, polypeptide, orantibody with a first array having a plurality of capture probes and asecond array having a different plurality of capture probes. The resultsof each hybridization can be compared, e.g., to analyze differences inexpression between a first and second sample. The first plurality ofcapture probes can be from a control sample, e.g., a wild type, normal,or non-diseased, non-stimulated, sample, e.g., a biological fluid,tissue, or cell sample. The second plurality of capture probes can befrom an experimental sample, e.g., a mutant type, at risk, disease-stateor disorder-state, or stimulated, sample, e.g., a biological fluid,tissue, or cell sample.

The plurality of capture probes can be a plurality of nucleic acidprobes each of which specifically hybridizes, with an allele of 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577.Such methods can be used to diagnose a subject, e.g., to evaluate riskfor a disease or disorder, to evaluate suitability of a selectedtreatment for a subject, to evaluate whether a subject has a disease ordisorder.

The method can be used to detect SNPs, as described above.

In another aspect, the invention features, a method of analyzing 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577,e.g., analyzing structure, function, or relatedness to other nucleicacid or amino acid sequences. The method includes: providing a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577nucleic acid or amino acid sequence; comparing the 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 sequence withone or more preferably a plurality of sequences from a collection ofsequences, e.g., a nucleic acid or protein sequence database; to therebyanalyze 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577.

The method can include evaluating the sequence identity between a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577sequence and a database sequence. The method can be performed byaccessing the database at a second site, e.g., over the internet.Preferred databases include GenBank™ and SwissProt.

In another aspect, the invention features, a set of oligonucleotides,useful, e.g., for identifying SNP's, or identifying specific alleles of27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577. The set includes a plurality of oligonucleotides, each of whichhas a different nucleotide at an interrogation position, e.g., an SNP orthe site of a mutation. In a preferred embodiment, the oligonucleotidesof the plurality identical in sequence with one another (except fordifferences in length). The oligonucleotides can be provided withdifferential labels, such that an oligonucleotide which hybridizes toone allele provides a signal that is distinguishable from anoligonucleotides which hybridizes to a second allele.

The sequences of 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 molecules are provided in a variety of mediums tofacilitate use thereof. A sequence can be provided as a manufacture,other than an isolated nucleic acid or amino acid molecule, whichcontains a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 molecule. Such a manufacture can provide anucleotide or amino acid sequence, e.g., an open reading frame, in aform which allows examination of the manufacture using means notdirectly applicable to examining the nucleotide or amino acid sequences,or a subset thereof, as they exist in nature or in purified form.

A 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 nucleotide or amino acid sequence can be recorded on computerreadable media. As used herein, “computer readable media” refers to anymedium that can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as compact disc and CD-ROM; electrical storage media such asRAM, ROM, EPROM, EEPROM, and the like; and general hard disks andhybrids of these categories such as magnetic/optical storage media. Themedium is adapted or configured for having thereon 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 sequenceinformation of the present invention.

As used herein, the term “electronic apparatus” is intended to includeany suitable computing or processing apparatus of other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as personal digital assistants(PDAs), cellular phones, pagers, and the like; and local and distributedprocessing systems.

As used herein, “recorded” refers to a process for storing or encodinginformation on the electronic apparatus readable medium. Those skilledin the art can readily adopt any of the presently known methods forrecording information on known media to generate manufactures comprisingthe 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552or 65577 sequence information.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon a 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577nucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order toobtain computer readable medium having recorded thereon the nucleotidesequence information of the present invention.

By providing the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 nucleotide or amino acid sequences of theinvention in computer readable form, the skilled artisan can routinelyaccess the sequence information for a variety of purposes. For example,one skilled in the art can use the nucleotide or amino acid sequences ofthe invention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. A search is used to identify fragments or regions ofthe sequences of the invention which match a particular target sequenceor target motif. The present invention therefore provides a medium forholding instructions for performing a method for determining whether asubject has a phospholipase, serine carboxypeptidase, trypsin-likeserine protease, aldehyde dehydrogenase, ubiquitin-protein ligase,protein kinase, hydrolase or matrix metalloproteinase-associated oranother 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577-associated disease or disorder or a pre-disposition to aphospholipase, serine carboxypeptidase, trypsin-like serine protease,aldehyde dehydrogenase, ubiquitin-protein ligase, protein kinase,hydrolase or matrix metalloproteinase-associated or another 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577-associated disease or disorder, wherein the method comprises thesteps of determining 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 sequence information associated with thesubject and based on the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 sequence information, determiningwhether the subject has a phospholipase, serine carboxypeptidase,trypsin-like serine protease, aldehyde dehydrogenase, ubiquitin-proteinligase, protein kinase, hydrolase or matrix metalloproteinase-associatedor another 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577-associated disease or disorder and/or recommendinga particular treatment for the disease, disorder, or pre-diseasecondition.

The present invention further provides in an electronic system and/or ina network, a method for determining whether a subject has aphospholipase, serine carboxypeptidase, trypsin-like serine protease,aldehyde dehydrogenase, ubiquitin-protein ligase, protein kinase,hydrolase or matrix metalloproteinase-associated or another 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577-associated disease or disorder or a pre-disposition to a diseaseassociated with 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577, wherein the method comprises the steps ofdetermining 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 sequence information associated with the subject,and based on the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 sequence information, determining whether thesubject has a phospholipase, serine carboxypeptidase, trypsin-likeserine protease, aldehyde dehydrogenase, ubiquitin-protein ligase,protein kinase, hydrolase or matrix metalloproteinase-associated oranother 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577-associated disease or disorder or a pre-disposition to aphospholipase, serine carboxypeptidase, trypsin-like serine protease,aldehyde dehydrogenase, ubiquitin-protein ligase, protein kinase,hydrolase or matrix metalloproteinase-associated or another 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577-associated disease or disorder, and/or recommending a particulartreatment for the disease, disorder, or pre-disease condition. Themethod may further comprise the step of receiving phenotypic informationassociated with the subject and/or acquiring from a network phenotypicinformation associated with the subject.

The present invention also provides in a network, a method fordetermining whether a subject has a phospholipase, serinecarboxypeptidase, trypsin-like serine protease, aldehyde dehydrogenase,ubiquitin-protein ligase, protein kinase, hydrolase or matrixmetalloproteinase-associated or another 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577-associated disease ordisorder or a pre-disposition to a phospholipase, serinecarboxypeptidase, trypsin-like serine protease, aldehyde dehydrogenase,ubiquitin-protein ligase, protein kinase, hydrolase or matrixmetalloproteinase-associated or another 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577-associated disease ordisorder, said method comprising the steps of receiving 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 sequenceinformation from the subject and/or information related thereto,receiving phenotypic information associated with the subject, acquiringinformation from the network corresponding to 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 and/orcorresponding to a phospholipase, serine carboxypeptidase, trypsin-likeserine protease, aldehyde dehydrogenase, ubiquitin-protein ligase,protein kinase, hydrolase or matrix metalloproteinase-associated oranother 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577-associated disease or disorder, and based on one or moreof the phenotypic information, the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 information (e.g., sequenceinformation and/or information related thereto), and the acquiredinformation, determining whether the subject has a phospholipase, serinecarboxypeptidase, trypsin-like serine protease, aldehyde dehydrogenase,ubiquitin-protein ligase, protein kinase, hydrolase or matrixmetalloproteinase-associated or another 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577-associated disease ordisorder or a pre-disposition to a phospholipase, serinecarboxypeptidase, trypsin-like serine protease, aldehyde dehydrogenase,ubiquitin-protein ligase, protein kinase, hydrolase or matrixmetalloproteinase-associated or another 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577-associated disease ordisorder. The method may further comprise the step of recommending aparticular treatment for the disease, disorder, or pre-diseasecondition.

The present invention also provides a business method for determiningwhether a subject has a phospholipase, serine carboxypeptidase,trypsin-like serine protease, aldehyde dehydrogenase, ubiquitin-proteinligase, protein kinase, hydrolase or matrix metalloproteinase-associatedor another 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577-associated disease or disorder or apre-disposition to a phospholipase, serine carboxypeptidase,trypsin-like serine protease, aldehyde dehydrogenase, ubiquitin-proteinligase, protein kinase, hydrolase or matrix metalloproteinase-associatedor another 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577-associated disease or disorder, said methodcomprising the steps of receiving information related to 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 (e.g.,sequence information and/or information related thereto), receivingphenotypic information associated with the subject, acquiringinformation from the network related to 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 and/or related to aphospholipase, serine carboxypeptidase, trypsin-like serine protease,aldehyde dehydrogenase, ubiquitin-protein ligase, protein kinase,hydrolase or matrix metalloproteinase-associated or another 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577-associated disease or disorder, and based on one or more of thephenotypic information, the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 information, and the acquiredinformation, determining whether the subject has a phospholipase, serinecarboxypeptidase, trypsin-like serine protease, aldehyde dehydrogenase,ubiquitin-protein ligase, protein kinase, hydrolase or matrixmetalloproteinase-associated or another 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577-associated disease ordisorder or a pre-disposition to a phospholipase, serinecarboxypeptidase, trypsin-like serine protease, aldehyde dehydrogenase,ubiquitin-protein ligase, protein kinase, hydrolase or matrixmetalloproteinase-associated or another 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577-associated disease ordisorder. The method may further comprise the step of recommending aparticular treatment for the disease, disorder, or pre-diseasecondition.

The invention also includes an array comprising a 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 sequence of thepresent invention. The array can be used to assay expression of one ormore genes in the array. In one embodiment, the array can be used toassay gene expression in a tissue to ascertain tissue specificity ofgenes in the array. In this manner, up to about 7600 genes can besimultaneously assayed for expression, one of which can be 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577. Thisallows a profile to be developed showing a battery of genes specificallyexpressed in one or more tissues.

In addition to such qualitative information, the invention allows thequantitation of gene expression. Thus, not only tissue specificity, butalso the level of expression of a battery of genes in the tissue ifascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionin that tissue. Thus, one tissue can be perturbed and the effect on geneexpression in a second tissue can be determined. In this context, theeffect of one cell type on another cell type in response to a biologicalstimulus can be determined. In this context, the effect of one cell typeon another cell type in response to a biological stimulus can bedetermined. Such a determination is useful, for example, to know theeffect of cell-cell interaction at the level of gene expression. If anagent is administered therapeutically to treat one cell type but has anundesirable effect on another cell type, the invention provides an assayto determine the molecular basis of the undesirable effect and thusprovides the opportunity to co-administer a counteracting agent orotherwise treat the undesired effect. Similarly, even within a singlecell type, undesirable biological effects can be determined at themolecular level. Thus, the effects of an agent on expression of otherthan the target gene can be ascertained and counteracted.

In another embodiment, the array can be used to monitor the time courseof expression of one or more genes in the array. This can occur invarious biological contexts, as disclosed herein, for exampledevelopment of a protein kinase, methyltransferase, acyl-CoAdehydrogenase, short chain dehyrdogenase, reductase, acyltransferase,phosphatase, transferase, ATP-ase or synthase-associated or another27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577-associated disease or disorder, progression of protein kinase,methyltransferase, acyl-CoA dehydrogenase, short chain dehyrdogenase,reductase, acyltransferase, phosphatase, transferase, ATP-ase orsynthase-associated or another 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577-associated disease or disorder, andprocesses, such a cellular transformation associated with thephospholipase, serine carboxypeptidase, trypsin-like serine protease,aldehyde dehydrogenase, ubiquitin-protein ligase, protein kinase,hydrolase or matrix metalloproteinase-associated or another 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577-associated disease or disorder.

The array is also useful for ascertaining the effect of the expressionof a gene on the expression of other genes in the same cell or indifferent cells (e.g., acertaining the effect of 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 expression onthe expression of other genes). This provides, for example, for aselection of alternate molecular targets for therapeutic intervention ifthe ultimate or downstream target cannot be regulated.

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes (e.g., including 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577) that could serve as amolecular target for diagnosis or therapeutic intervention.

As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. Typical sequence lengths of a targetsequence are from about 10 to 100 amino acids or from about 30 to 300nucleotide residues. However, it is well recognized that commerciallyimportant fragments, such as sequence fragments involved in geneexpression and protein processing, may be of shorter length.

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable mediumfor analysis and comparison to other sequences. A variety of knownalgorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware include, but are not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBI).

Thus, the invention features a method of making a computer readablerecord of a sequence of a 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 sequence which includes recordingthe sequence on a computer readable matrix. In a preferred embodimentthe record includes one or more of the following: identification of anORF; identification of a domain, region, or site; identification of thestart of transcription; identification of the transcription terminator;the full length amino acid sequence of the protein, or a mature formthereof; the 5′ end of the translated region.

In another aspect, the invention features a method of analyzing asequence. The method includes: providing a 27877, 18080, 14081, 32140,50352, 16658, 14223, 16002, 50566, 65552 or 65577 sequence, or record,in computer readable form; comparing a second sequence to the 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577sequence; thereby analyzing a sequence. Comparison can include comparingto sequences for sequence identity or determining if one sequence isincluded within the other, e.g., determining if the 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 sequenceincludes a sequence being compared. In a preferred embodiment the 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577or second sequence is stored on a first computer, e.g., at a first siteand the comparison is performed, read, or recorded on a second computer,e.g., at a second site. E.g., the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 or second sequence can bestored in a public or proprietary database in one computer, and theresults of the comparison performed, read, or recorded on a secondcomputer. In a preferred embodiment the record includes one or more ofthe following: identification of an ORF; identification of a domain,region, or site; identification of the start of transcription;identification of the transcription terminator; the full length aminoacid sequence of the protein, or a mature form thereof; the 5′ end ofthe translated region.

EXEMPLIFICATION Example 1 Tissue Distribution of 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 mRNA

Northern blot hybridizations with various RNA samples can be performedunder standard conditions and washed under stringent conditions, i.e.,0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 cDNA (SEQ ID NO:1, 3, 4, 6, 11, 13, 20, 22, 25, 27, 29, 31, 35,37, 38, 40, 41, 43, 73, 75, 76, 78, 86 or 88) or 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 cDNA can beused. The DNA was radioactively labeled with ³²P-dCTP using the Prime-ItKit (Stratagene, La Jolla, Calif.) according to the instructions of thesupplier. Filters containing mRNA from mouse hematopoietic and endocrinetissues, and cancer cell lines (Clontech, Palo Alto, Calif.) can beprobed in ExpressHyb hybridization solution (Clontech) and washed athigh stringency according to manufacturer's recommendations.

Example 2 Recombinant Expression of 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 in Bacterial Cells

In this example, 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577is fused to GST and this fusion polypeptide is expressed in E. coli,e.g., strain PEB199. Expression of the GST-27877, -18080, -14081,-32140, -50352, -16658, -14223, -16002, -50566, -65552 or -65577 fusionprotein in PEB199 is induced with IPTG. The recombinant fusionpolypeptide is purified from crude bacterial lysates of the inducedPEB199 strain by affinity chromatography on glutathione beads. Usingpolyacrylamide gel electrophoretic analysis of the polypeptide purifiedfrom the bacterial lysates, the molecular weight of the resultant fusionpolypeptide is determined.

Example 3 Expression of Recombinant 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 Protein in COS Cells

To express the 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002,50566, 65552 or 65577 gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 protein and an HA tag (Wilson et al. (1984) Cell 37:767)or a FLAG tag fused in-frame to its 3′ end of the fragment is clonedinto the polylinker region of the vector, thereby placing the expressionof the recombinant protein under the control of the CMV promoter.

To construct the plasmid, the 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577coding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the 27877, 18080, 14081, 32140, 50352, 16658, 14223,16002, 50566, 65552 or 65577 coding sequence. The PCR amplified fragmentand the pcDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 gene is inserted in thecorrect orientation. The ligation mixture is transformed into E. colicells (strains HB101, DH5α, SURE, available from Stratagene CloningSystems, La Jolla, Calif., can be used), the transformed culture isplated on ampicillin media plates, and resistant colonies are selected.Plasmid DNA is isolated from transformants and examined by restrictionanalysis for the presence of the correct fragment.

COS cells are subsequently transfected with the 27877-, 18080-, 14081-,32140-, 50352-, 16658-, 14223-, 16002-, 50566-, 65552- or65577-pcDNA/Amp plasmid DNA using the calcium phosphate or calciumchloride co-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 polypeptide is detected byradiolabelling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold-Spring Harbor, N.Y., 1988) using an HA specific monoclonalantibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine(or ³⁵S-cysteine). The culture media are then collected and the cellsare lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1%SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA specific monoclonal antibody.Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577 coding sequence is cloneddirectly into the polylinker of the pcDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or65577 polypeptide is detected by radiolabelling and immunoprecipitationusing a 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 specific monoclonal antibody.

Exmaple 4 TaqMan Analysis of 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577

Human 27877, 18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566,65552 or 65577 expression was measured by TaqMan® quantitative PCR(Perkin Elmer Applied Biosystems) in cDNA prepared from a variety ofnormal and diseased (e.g., cancerous) human tissues or cell lines.

Probes were designed by PrimerExpress software (PE Biosystems) based onthe sequence of the human 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 gene. Each human 27877, 18080,14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 geneprobe was labeled using FAM (6-carboxyfluorescein), and theβ2-microglobulin reference probe was labeled with a differentfluorescent dye, VIC. The differential labeling of the target gene andinternal reference gene thus enabled measurement in same well. Forwardand reverse primers and the probes for both β2-microglobulin and targetgene were added to the TaqMan® Universal PCR Master Mix (PE AppliedBiosystems). Although the final concentration of primer and probe couldvary, each was internally consistent within a given experiment. Atypical experiment contained 200 nM of forward and reverse primers plus100 nM probe for β-2 microglobulin and 600 nM forward and reverseprimers plus 200 nM probe for the target gene. TaqMan matrix experimentswere carried out on an ABI PRISM 7700 Sequence Detection System (PEApplied Biosystems). The thermal cycler conditions were as follows: holdfor 2 min at 50° C. and 10 min at 95° C., followed by two-step PCR for40 cycles of 95° C. for 15 sec followed by 60° C. for 1 min.

The following method was used to quantitatively calculate human 27877,18080, 14081, 32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577gene expression in the various tissues relative to β-2 microglobulinexpression in the same tissue. The threshold cycle (Ct) value is definedas the cycle at which a statistically significant increase influorescence is detected. A lower Ct value is indicative of a highermRNA concentration. The Ct value of the human 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 gene isnormalized by subtracting the Ct value of the β-2 microglobulin gene toobtain a _(Δ)Ct value using the following formula:_(Δ)Ct=Ct_(human 59914 and 59921)−Ct_(β-2 microglobulin). Expression isthen calibrated against a cDNA sample showing a comparatively low levelof expression of the human 27877, 18080, 14081, 32140, 50352, 16658,14223, 16002, 50566, 65552 or 65577 gene. The _(Δ)Ct value for thecalibrator sample is then subtracted from _(Δ)Ct for each tissue sampleaccording to the following formula:_(ΔΔ)Ct=_(Δ)Ct-_(sample)−_(Δ)Ct-_(calibrator). Relative expression isthen calculated using the arithmetic formula given by 2^(−ΔΔCt).

Example 5 In Situ Hybridization of 27877, 18080, 14081, 32140, 50352,16658, 14223, 16002, 50566, 65552 or 65577

The following describes the tissue distribution of 27877, 18080, 14081,32140, 50352, 16658, 14223, 16002, 50566, 65552 or 65577 mRNA, as may bedetermined by in situ hybridization analysis using oligonucleotideprobes based on the human G2RF sequence.

For in situ analysis, various tissues, e.g. tissues obtained from brain,are first frozen on dry ice. Ten-micrometer-thick sections of thetissues are postfixed with 4% formaldehyde in DEPC treated 1×phosphate-buffered saline at room temperature for 10 minutes beforebeing rinsed twice in DEPC 1× phosphate-buffered saline and once in 0.1M triethanolamine-HCl (pH 8.0). Following incubation in 0.25% aceticanhydride-0.1 M triethanolamine-HCl for 10 minutes, sections are rinsedin DEPC 2×SSC (1×SSC is 0.15M NaCl plus 0.015M sodium citrate). Tissueis then dehydrated through a series of ethanol washes, incubated in 100%chloroform for 5 minutes, and then rinsed in 100% ethanol for 1 minuteand 95% ethanol for 1 minute and allowed to air dry.

Hybridizations are performed with ³⁵S-radiolabeled (5×10⁷ cpm/ml) cRNAprobes. Probes are incubated in the presence of a solution containing600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon spermDNA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1, 1× Denhardt'ssolution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol,0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18hours at 55° C.

After hybridization, slides are washed with 2×SSC. Sections are thensequentially incubated at 37° C. in TNE (a solution containing 10 mMTris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNEwith 10 μg of RNase A per ml for 30 minutes, and finally in TNE for 10minutes. Slides are then rinsed with 2×SSC at room temperature, washedwith 2×SSC at 50° C. for 1 hour, washed with 0.2×SSC at 55° C. for 1hour, and 0.2×SSC at 60° C. for 1 hour. Sections are then dehydratedrapidly through serial ethanol-0.3 M sodium acetate concentrationsbefore being air dried and exposed to Kodak Biomax MR scientific imagingfilm for 24 hours and subsequently dipped in NB-2 photoemulsion andexposed at 4° C. for 7 days before being developed and counter stained.

The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein.

1. An isolated nucleic acid selected from the group consisting of: a) anucleic acid comprising the nucleotide sequence of SEQ ID NO:41 or SEQID NO:43; b) a nucleic acid which encodes a polypeptide comprising theamino acid sequence of SEQ ID NO:42; c) a nucleic acid comprising anucleotide sequence which is at least 95% identical to the nucleotidesequence of SEQ ID NO:41 or SEQ ID NO:43, wherein the nucleotidesequence encodes a polypeptide which has calcium/calmodulin-dependentprotein kinase kinase alpha activity; and d) a nucleic acid whichencodes a polypeptide comprising an amino acid sequence which is atleast 95% identical to the amino acid sequence of SEQ ID NO:42, whereinthe polypeptide has calcium/calmodulin-dependent protein kinase kinasealpha activity.
 2. The nucleic acid of claim 1, wherein the nucleic acidencodes a polypeptide comprising an amino acid sequence which is atleast 95% identical to the amino acid sequence of SEQ ID NO:42, whereinthe polypeptide has calcium/calmodulin-dependent protein kinase kinasealpha activity.
 3. The nucleic acid of claim 1, wherein the nucleic acidcomprises a nucleotide sequence which is at least 95% identical to thenucleotide sequence of SEQ ID NO:41 or SEQ ID NO:43, wherein thenucleotide sequence encodes a polypeptide which hascalcium/calmodulin-dependent protein kinase kinase alpha activity. 4.The nucleic acid of claim 1, wherein the nucleic acid comprises thenucleotide sequence of SEQ ID NO:41 or SEQ ID NO:43.
 5. The nucleic acidof claim 1, wherein the nucleic acid encodes a polypeptide comprisingthe amino acid sequence of SEQ ID NO:
 42. 6. The nucleic acid of claim1, wherein the nucleic acid consists of the nucleotide sequence of SEQID NO:41 or SEQ ID NO:43.
 7. The nucleic acid of claim 1, wherein thenucleic acid consists of a nucleotide sequence which encodes the aminoacid sequence of SEQ ID NO:
 42. 8. A vector comprising the nucleic acidof claim
 1. 9. A vector comprising the nucleic acid of claim
 2. 10. Avector comprising the nucleic acid of claim
 3. 11. An isolated host cellcomprising the nucleic acid of claim
 1. 12. An isolated host cellcomprising the nucleic acid of claim
 2. 13. An isolated host cellcomprising the nucleic acid of claim
 3. 14. A method for producing apolypeptide selected from the group consisting of: a) a polypeptidecomprising the amino acid sequence of SEQ ID NO:42; b) a polypeptideencoded by a nucleic acid comprising the nucleotide sequence of SEQ IDNO:41 or SEQ ID NO:43; c) a polypeptide comprising an amino acidsequence which is at least 95% identical to the amino acid sequence ofSEQ ID NO:42, wherein the polypeptide has calcium/calmodulin-dependentprotein kinase kinase alpha activity; and d) a polypeptide encoded by anucleic acid comprising a nucleotide sequence which is at least 95%identical to the nucleotide sequence of SEQ ID NO:41 or SEQ ID NO:43,wherein the polypeptide has calcium/calmodulin-dependent protein kinasekinase alpha activity; the method comprising culturing the host cell ofclaim 11 under conditions in which the polypeptide is expressed andthereby producing the polypeptide.